Biomolecule substrate, and test and diagnosis methods and apparatuses using the same

ABSTRACT

An object of the present invention is to provide a test apparatus for testing a DNA substrate on which a plurality of DNA fragments for testing are arranged, wherein absolute precision is not required. The above-described problem was solved by providing a substrate on which a plurality of biomolecule spots containing a group of biomolecules (e.g., DNA, etc.) of a specific type are formed, where the pattern or position of the DNA spot is changed depending on specific data so that information of the specific data is recorded on the substrate.

This Application is a U.S. National Phase Application of PCTInternational Application PCT/JP02/04597.

TECHNICAL FIELD

The present invention relates to a substrate for use in a test fordetecting a biomolecule (e.g., DNA, RNA, a protein, a low-weight organicmolecule (ligand, etc.), sugar, lipid, etc.), a biomolecule chip, and adetection apparatus and test (including screening) and diagnosis methodsusing the same.

BACKGROUND ART

Recently, science and technologies related to genes have been developedmore remarkably than expected. As a technique for detecting, analyzingand measuring genetic information, an apparatus called a biomoleculechip (including a DNA chip, a biochip, a microarray, a protein chip,etc.) and a test method using the same have recently received attention.A number of different nucleic acids (DNA such as cDNA and genomic DNA,RNA, PNA, etc.) or peptides are arranged and fixed in spotted pattern ona substrate made of glass or silicon. On this substrate, fragments ofsample DNA to be tested are hybridized with a labeling substance, suchas a fluorophore or an isotope or the like, and capture DNA, oralternatively, a sample polypeptide or ligand to be tested is conjugatedwith a labeling protein by means of their interaction. A detector isused to detect fluorescence from the labeled DNA or the labeling peptidein each spot, or a radiation detector is used to detect radioactivitytherefrom, thereby obtaining information on arrangement of labeled DNAor labeling peptide spots. By analyzing this data, genetic informationon the sample DNA can be obtained.

A gene detection method using a DNA chip or the like has the potentialto be widely used in the analysis of genes for the diagnosis of adisease or analysis of an organism in the future. Examples of a chipapplication include screening of a compound library for combinatorialchemistry or the like. The versatility of the chips also has receivedattention.

To date, however, methods for fabricating biomolecule chips as describedabove require high-precision equipment, leading to high cost for adetection substrate. Moreover, an apparatus for detecting a labeled DNArequires high precision, and therefore, it is difficult for such anapparatus to come into widespread use in small business entities orpractitioners. Biomolecule chips do not have sufficient ability toprocess a large amount of data. Therefore, a substrate or a chip capableof processing data in an easy and efficient manner is expected.

Problems to be Solved by the Invention

The above-described detection substrate or detection apparatus demands amethod which does not require high precision. An object of the presentinvention is to provide a system which can be made even using apoor-precision test apparatus and in which system a test can beperformed.

DISCLOSURE OF THE INVENTION

To solve the above-described problem, the present invention provides anapparatus comprising a substrate on which a plurality of biomoleculespots made of a specific type of biomolecule (e.g., DNA, etc.), in whicha pattern or arrangement of the spot of the biomolecule (e.g., DNA) ischanged depending on specific data so that the data is recorded on thesubstrate.

Therefore, the present invention provides the following.

In one aspect, the present invention provides a method for fabricating abiomolecule substrate, comprising the steps of: 1) providing a set ofbiomolecules and a substrate; 2) enclosing the set of biomolecules intomicrocapsules on the biomolecule-type-by-biomolecule-type basis; and 3)spraying the biomolecule microcapsules onto the substrate.

In one embodiment, the present invention further comprises the step ofwashing the biomolecule microcapsules after the enclosing step.

In another embodiment, the spraying step is performed by an ink jetmethod.

In another embodiment, the ink jet method is performed by a BUBBLE JET®method.

In another embodiment, the present invention further comprises the stepof setting the temperature of a solution used in the spraying step to behigher than the melting point of a shell of the biomoleculemicrocapsulate.

In another embodiment, the microcapsules of the set of biomolecules ofdifferent types are disposed at different positions.

In another embodiment, the spraying step is performed by a PIN method.

In another embodiment, the biomolecule contains at least one of DNA, RNAand a peptide.

In another embodiment, the biomolecule is DNA.

In another embodiment, the biomolecule is cDNA or genomic DNA.

In another embodiment, the present invention further comprises the stepof perform labeling specific to each microcapsule.

In another aspect, the present invention provides a biomolecule chip,comprising: a substrate; and biomolecules and chip attribute dataarranged on the substrate, wherein the chip attribute data is arrangedin the same region as that of the biomolecules.

In one embodiment, the chip attribute data contains information relatingto chip ID and the substrate.

In another embodiment, the present invention further comprises arecording region, wherein the recording region is placed on the samesubstrate as that of the biomolecule and the chip attribute data, and atleast one of subject data and measurement data is recorded in therecording region.

In another embodiment, the chip attribute data is recorded in such amanner as to be read out by the same means as that for detecting thebiomolecule.

In another embodiment, a specific mark is attached to the substrate.

In another embodiment, a specific mark is arranged based on the chipattribute data.

In another embodiment, the chip attribute data contains the biomoleculeattribute data.

In another embodiment, information relating to an address of thebiomolecule is further recorded.

In another embodiment, the address is a tracking address.

In another embodiment, the chip attribute data is encrypted.

In another embodiment, data relating to a label used to detect thebiomolecule is recorded.

In another embodiment, the data relating to the label contains at leastone of the wavelength of excited light and the wavelength offluorescence.

In another embodiment, the biomolecule contains at least one of DNA, RNAand a peptide.

In another embodiment, the biomolecule is DNA.

In another embodiment, the biomolecule is cDNA or genomic DNA.

In another aspect, the present invention provides a biomolecule chip,comprising: 1) a substrate; and 2) biomolecules arranged on thesubstrate, wherein spots of the biomolecules are spaced by at least onenon-equal interval, an address of the biomolecule spot can be identifiedfrom the non-equal interval.

In one embodiment, the non-equal interval is modulated.

In another embodiment, the non-equal interval is present in at least twodirections.

In another aspect, the present invention provides a biomolecule chip.This biomolecule chip comprises: 1) a substrate; and 2) biomoleculesarranged on the substrate, wherein the biomolecules include adistinguishable first biomolecule and a distinguishable secondbiomolecule, an address of the biomolecule can be identified based on anarrangement of spots of the first biomolecules and spots of the secondbiomolecule.

In one embodiment, a label distinguishable from the biomolecule isplaced between the biomolecule spots.

In another embodiment, the distinguishable label can be detected bydetection means.

In another embodiment, the label is arranged in a horizontal directionand a vertical direction on the substrate.

In another embodiment, a synchronization mark is arranged.

In another embodiment, the biomolecule contains at least one of DNA, RNAand a peptide.

In another embodiment, the biomolecule is DNA.

In another embodiment, the biomolecule is cDNA or genomic DNA.

In another aspect, the present invention provides a biomolecule chip,comprising: 1) a substrate; and 2) biomolecules arranged on thesubstrate, wherein spots storing attribute data are arranged on a sideof the substrate opposite to a side on which spots of the biomoleculesare arranged.

In another embodiment, the attribute data is address information.

In another aspect, the present invention provides a biomolecule chip,comprising: 1) a substrate; 2) biomolecules arranged on the substrate;and 3) a data recording region.

In one embodiment, the data recording region is placed on the sideopposite to the side on which the biomolecules are arranged.

In another aspect, the present invention provides a method for detectinga label of a biomolecule chip, comprising the steps of: 1) providing abiomolecule chip on which at least one labeled biomolecule is arranged;2) switching detection elements sequentially for detecting thebiomolecules on the biomolecule chip; and 3) identifying a signaldetected by the detection element.

In one embodiment, the present invention further comprises: 4) adding upeach detected signal.

In another embodiment, the signal is separated by a wavelengthseparation mirror.

In another embodiment, the biomolecule substrate further contains asynchronization mark, and the label is identified based on thesynchronization mark.

In another embodiment, the biomolecule substrate contains addressinformation on a rear side of the biomolecule, and the label isidentified based on the address information.

In another aspect, the present invention provides a method for detectinginformation on an organism, comprising the steps of: 1) providing abiomolecule sample from the organism; 2) providing the biomolecule chipof the present invention; 3) contacting the biomolecule sample to thebiomolecule chip, and placing the biomolecule chip under conditionswhich causes an interaction between the biomolecule sample and abiomolecule placed on the biomolecule; and 4) detecting a signal causedby the biomolecule and a signal caused by the interaction, wherein thesignal is an indicator for at least one information parameter of theorganism, and the signal is related to an address assigned to thenon-equal interval or the spot arrangement.

In another embodiment, the biomolecule sample contains nucleic acid, andthe biomolecule placed on the biomolecule chip is nucleic acid.

In another embodiment, the sample contains a protein and the biomoleculeplaced on the biomolecule chip is an antibody, or the sample contains anantibody and the biomolecule placed on the biomolecule chip is aprotein.

In another embodiment, the present invention further comprises labelingthe biomolecule sample with a label molecule.

In another embodiment, the label molecule can be distinguished from thebiomolecule placed on the biomolecule chip.

In another embodiment, the label molecule contains a fluorescentmolecule, a photophorescent molecule, a chemoluminescent molecule, or aradioactive isotope.

In another embodiment, the signal detecting step is performed at a sitedifferent from where the interaction occurs.

In another embodiment, the signal detecting step is performed at thesame site as where the interaction occurs.

In another embodiment, the present invention further comprisesencrypting the signal.

In another embodiment, the present invention further comprisessubjecting the signal to filtering so as to extract only signal relatingto required information.

In another aspect, the present invention provides a method fordiagnosing a subject, comprising the steps of: 1) providing a samplefrom the subject; 2) providing the biomolecule chip of the presentinvention; 3) contacting the biomolecule sample to the biomolecule chip,and placing the biomolecule chip under conditions which cause aninteraction between the biomolecule sample and a biomolecule placed onthe biomolecule; 4) detecting a signal caused by the biomolecule and asignal caused by the interaction, wherein the signal is at least onediagnostic indicator for the subject, and the signal is related to anaddress assigned to the non-equal interval or the spot arrangement; and5) determining the diagnostic indicator from the signal.

In another embodiment, the sample is nucleic acid, and the biomoleculeplaced on the biomolecule chip is nucleic acid.

In another embodiment, the sample contains a protein and the biomoleculeplaced on the biomolecule chip is an antibody, or the sample contains anantibody and the biomolecule placed on the biomolecule chip is aprotein.

In another embodiment, the present invention further comprises labelingthe sample with a label molecule.

In another embodiment, the label molecule can be distinguished from thebiomolecule placed on the biomolecule chip.

In another embodiment, the label molecule is a fluorescence molecule, aphotophorescent molecule, a chemoluminescent molecule, or a radioactiveisotope.

In another embodiment, the diagnostic indicator is an indicator for adisease or a disorder.

In another embodiment, the diagnostic indicator is based on singlenucleotide polymorphism (SNP).

In another embodiment, the diagnostic indicator is based on a geneticdisease.

In another embodiment, the diagnostic indicator is based on theexpression level of a protein.

In another embodiment, the diagnostic indicator is based on a testresult of a biochemical test.

In another embodiment, the determining step is performed at a sitedifferent from where the interaction occurs.

In another embodiment, the signal detecting step is performed at thesame site as where the interaction occurs.

In another embodiment, the present invention further comprisesencrypting the signal.

In another embodiment, the present invention further comprisessubjecting the signal to filtering so as to extract only signal relatingto required information.

In another embodiment, in the detecting step biomolecule attribute datais hidden, and in the determining step personal information data ishidden.

In another aspect, the present invention provides a test apparatus forinformation on an organism, comprising: 1) the biomolecule chip of thepresent invention; 2) a sample applying section in fluid communicationwith the biomolecule chip; 3) a reaction control section for controllinga contact and an interaction between the biomolecule placed on thebiomolecule and a biomolecule sample applied from the sample applyingsection; and 4) a detection section for detecting a signal caused by theinteraction, wherein the signal is an indicator for at least oneinformation parameter of the organism, and the signal is related to anaddress assigned to the non-equal interval or the spot arrangement.

In another embodiment, the present invention further comprises a sectionfor receiving and sending the signal.

In another embodiment, the present invention further comprises a regionfor recording the signal.

In another aspect, the present invention provides a diagnosis apparatusfor a subject. This diagnosis apparatus comprisies: 1) the biomoleculechip of the present invention; 2) a sample applying section in fluidcommunication with the biomolecule chip; 3) a reaction control sectionfor controlling a contact and an interaction between the biomoleculeplaced on the biomolecule and a biomolecule sample applied from thesample applying section; 4) a detection section for detecting a signalcaused by the biomolecule and a signal caused by the interaction,wherein the signal is an indicator for at least one informationparameter of the organism, and the signal is related to an addressassigned to the non-equal interval or the spot arrangement; and 5)determining the diagnostic indicator from the signal.

In one embodiment, the present invention further comprises a section forreceiving and sending the signal.

In another embodiment, the present invention further comprises a regionfor recording the signal.

In one aspect, the present invention provides a biological test system.This biological test system comprises: A) a main sub system,comprising: 1) the biomolecule chip of the present invention; 2) asample applying section in fluid communication with the biomoleculechip; 3) a reaction control section for controlling a contact and aninteraction between the biomolecule placed on the biomolecule and abiomolecule sample applied from the sample applying section; 4) adetection section for detecting a signal caused by the biomolecule and asignal caused by the interaction, wherein the signal is an indicator forat least one information parameter of the organism, and the signal isrelated to an address assigned to the non-equal interval or the spotarrangement; and 5) a sending and receiving section for sending andreceiving a signal, and B) a sub sub system, comprising: 1) a sendingand receiving section for sending and receiving a signal; and 2) a testsection for calculating a test value from the signal received from themain sub system. The main sub system and the sub sub system areconnected together via a network.

In another embodiment, the signal received by the sub sub systemcontains a signal relating to measurement data measured by the sub subsystem.

In another embodiment, the attribute data contains chip ID, personalinformation data, and biomolecule attribute data, the main sub systemcontains the chip ID and the personal information data, but does notcontain the biomolecule attribute data, and the sub sub system containsthe chip ID and the biomolecule attribute data, but does not contain thepersonal information data, and the sub sub system sends the test value,determined in response to a request, to the main sub system.

In another embodiment, the network is the Internet.

In another embodiment, the signal to be sent and received is encrypted.

In another aspect, the present invention provides a diagnosis system.This diagnosis system comprises: A) a main sub system, comprising: 1)the biomolecule chip of the present invention; 2) a sample applyingsection in fluid communication with the biomolecule chip; 3) a reactioncontrol section for controlling a contact and an interaction between thebiomolecule placed on the biomolecule and a biomolecule sample appliedfrom the sample applying section; 4) a detection section for detecting asignal caused by the biomolecule and a signal caused by the interaction,wherein the signal is an indicator for at least one informationparameter of the organism, and the signal is related to an addressassigned to the non-equal interval or the spot arrangement; and 5) asending and receiving section for sending and receiving a signal, and B)a sub sub system, comprising: 1) a sending and receiving section forsending and receiving a signal; and 2) a determination section fordetermining the diagnostic indicator from the signal received from themain sub system. The main sub system and the sub sub system areconnected together via a network.

In another embodiment, the signal received by the sub sub systemcontains a signal relating to measurement data measured by the sub subsystem.

In another embodiment, the attribute data contains chip ID, personalinformation data, and biomolecule attribute data, the main sub systemcontains the chip ID and the personal information data, but does notcontain the biomolecule attribute data, and the sub sub system containsthe chip ID and the biomolecule attribute data, and data for determininga diagnostic indicator from biomolecule attribute data, but does notcontain the personal information data, and the sub sub system sends thediagnostic indicator, determined in response to a request, to the mainsub system.

In another embodiment, the network is the Internet.

In another embodiment, the signal to be sent and received is encrypted.

In another embodiment, the present invention provides a test apparatusfor biological information. This test apparatus comprises: a support fora substrate; a plurality of groups of biomolecules arranged on thesubstrate, each group containining the biomolecules of the same type;shifting means for shifting the substrate; a light source for exciting afluorescence substance labeling a sample to be tested; and optical meansfor converging light from the light source. The light source is causedto emit light intermittently in response to an intermittent emissionsignal so as to excite the fluorescence substance, fluorescence from thefluorescence substance is detected by a photodetector during a period oftime when the intermittent emission signal is paused, identificationinformation is reproduced from an arrangement of the DNAs, and thebiomolecules emitting fluorescence is identified.

In another embodiment, the present invention further comprises means foradding up detected detection signals.

In another embodiment, the present invention further comprises awavelength separation mirror.

In another embodiment, the present invention provides use of thebiomolecule chip of the present invention for fabricating an apparatusfor testing biological information.

In another embodiment, the present invention provides use of thebiomolecule chip of the present invention for fabricating an apparatusfor diagnosing a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be herein described with reference to thedrawings briefly described below. The drawings are provided for thepurpose of illustrating preferable embodiments of the present invention,but not for the purpose of restricting the scope of the presentinvention. The scope of the present invention is specified only by theclaims attached thereto. Each figure will be described below.

FIG. 1:

(a) A top view showing a substrate on which DNA is placed, according toan embodiment of the present invention.

(b) A cross-sectional view showing a substrate on which DNA is placed,according to an embodiment of the present invention.

FIG. 2:

A diagram showing a method for fabricating a DNA microcapsule accordingto an embodiment of the present invention.

FIG. 3:

A diagram shown in a method for attaching DNA by a pin method accordingto an embodiment of the present invention.

FIG. 4:

A diagram showing a method for shifting DNA to a pin according to anembodiment of the present invention.

FIG. 5:

A top view showing a DNA chip according to an embodiment of the presentinvention, and a data structure diagram.

FIG. 6:

A diagram showing a structure of DNA substrate attribute data accordingto an embodiment of the present invention.

FIG. 7:

A diagram showing a method for fixing DNA according to an embodiment ofthe present invention.

FIG. 8:

A schematic diagram showing a method for fixing DNA according to anembodiment of the present invention.

FIG. 9:

A block diagram showing a method for ejecting DNA by an ink jet methodaccording to an embodiment of the present invention.

FIG. 10:

A diagram showing an arrangement of DNA on a substrate according to anembodiment of the present invention.

FIG. 11:

A diagram showing ejection in an ink jet method according to anembodiment of the present invention.

FIG. 12:

A diagram showing an arrangement of DNA spots on a substrate accordingto an embodiment of the present invention.

FIG. 13:

A diagram showing hybridization of labeled DNA according to anembodiment of the present invention.

FIG. 14:

A block diagram showing a test apparatus according to an embodiment ofthe present invention.

FIG. 15:

A flowchart showing ejection of a microcapsule according to anembodiment of the present invention.

FIG. 16:

A diagram showing an operation of a mirror according to an embodiment ofthe present invention.

FIG. 17:

A diagram showing a relationship between excited light and fluorescenceaccording to an embodiment of the present invention.

FIG. 18:

A diagram showing scanning of DNA spots according to an embodiment ofthe present invention.

FIG. 19:

A diagram showing a relationship between a light receiving array andfluorescence according to an embodiment of the present invention.

FIG. 20:

A timing chart showing detection of fluorescence according to anembodiment of the present invention.

FIG. 21:

A block diagram showing a photodetector comprising a light receivingarray according to an embodiment of the present invention.

FIG. 22:

A diagram showing exemplary data of a label detection signal accordingto an embodiment of the present invention.

FIG. 23:

A diagram showing a principle of a detection apparatus according to anembodiment of the present invention.

FIG. 24:

A diagram showing a principle of a detection apparatus according to anembodiment of the present invention.

FIG. 25:

A top view showing a relationship between a DNA spot and a trackaccording to an embodiment of the present invention.

FIG. 26:

A diagram showing an arrangement of DNA spots according to an embodimentof the present invention.

FIG. 27:

A top view showing a circular substrate according to an embodiment ofthe present invention.

FIG. 28:

A diagram showing a DNA area of a circular substrate according to anembodiment of the present invention.

FIG. 29:

A diagram showing a procedure for fabricating a DNA substrate using asemiconductor process method according to an embodiment of the presentinvention.

FIG. 30:

A diagram showing a principle of an ink jet method according to anembodiment of the present invention.

FIG. 31:

A flowchart showing a method for detecting fluorescence by scanning aplurality of times according to an embodiment of the present invention.

FIG. 32:

A timing chart showing excited light and detected light in a method forscanning a plurality of times according to an embodiment of the presentinvention.

FIG. 33:

A diagram showing a method for fabricating a biomolecule chip by a tubemethod according to an embodiment of the present invention.

FIG. 34:

A diagram showing another method for fabricating a biomolecule chip by atube method according to an embodiment of the present invention.

FIG. 35:

A diagram showing an arrangement of biomolecule spots by a tube methodaccording to an embodiment of the present invention and a diagramshowing buried data.

FIG. 36:

A diagram showing an arrangement of biomolecule spots by a tube methodaccording to an embodiment of the present invention and a diagramshowing buried data.

FIG. 37:

A diagram showing an arrangement of biomolecule spots by a tube methodaccording to an embodiment of the present invention.

FIG. 38:

A diagram showing a method for arranging a biomolecule spot by a pinmethod according to an embodiment of the present invention.

FIG. 39:

A diagram showing a method for arranging a biomolecule spot by an inkjet method according to an embodiment of the present invention.

FIG. 40:

A diagram showing a table of an identification number and a biomoleculeattribute data according to an embodiment of the present invention.

FIG. 41:

A flowchart showing a detection procedure using a pin method accordingto an embodiment of the present invention.

FIG. 42:

A diagram showing a data structure of data containing ECC buried by atube method according to an embodiment of the present invention.

FIG. 43:

A block diagram showing a network type test system according to anembodiment of the present invention.

FIG. 44:

A block diagram showing a stand-alone type test system according to anembodiment of the present invention.

FIG. 45:

A diagram showing a table of analysis results according to an embodimentof the present invention.

FIG. 46:

A diagram showing a structure of a biomolecule chip according to anembodiment of the present invention.

FIG. 47:

A diagram showing a structure according to an embodiment of the presentinvention, in which an address can be specified by a specificarrangement.

FIG. 48:

A diagram showing a structure of a biomolecule chip according to anembodiment of the present invention, in which an address can bespecified by a specific pattern.

DESCRIPTION OF REFERENCE NUMERALS

1 substrate

2 DNA spot

3 DNA

4 main solution

5 main film

6 DNA microcapsule

7 sub-film

8 sub-solution

9 microcapsule

10 main container

11 container

12 tray

13 pin

14 moving pin

15 washing section

16 pin drum

17 DNA spot region

18 data region

19 substrate ID

20 DNA number-position correspondence table

21 DNA sequence data

22 labeled DNA

23 empty microcapsule

24 nozzle

25 supply section

26 eject section (heater)

27 eject control circuit

28 master control section

29 eject signal generation section

30 removal signal generation section

31 photodetector

32 unnecessary liquid removing section

33 deviation section

34 arrow

35 shift amount detector

36 shift control circuit

37 synchronization mark

38 fluorescence dye

39 detection apparatus

40 light source (for excitation)

41 mirror

42 lens

43 detection section

44 focus error signal detection section

45 tracking error signal detection section

46 focus control circuit

47 tracking control circuit

48 actuator

49 focus offset signal generation section

50 track offset signal generation section

51 spot number output section

52 track number output section

53 ECC decoder

54 DNA substrate attribute data reading portion

55 data processing section

56 synchronization signal generation section

57 substrate shift section

58 capture DNA number

59 second label signal detection section

60 first label signal detection section

61 first label signal output section

62 second label signal output section

63 data output section

64 positional information detection section

65 mirror

66 mirror

67 label signal detection section

68 step

69 main signal reproduction section

70 detection cell

71 excitation beam

72 scanning track

73 encryption key

74 cipher decoder

75 factory-shipped data region

76 postscript data region

77 first label attribute data

78 second label attribute data

79 synchronization data

80 data reproduction area

85 label detection signal

86 shift amount detector

87 pulsed light emission control section

88 pulsed light emission signal

89 sub-pulsed light emission signal

90 light detection section

91 array

92 switching section

93 addition section

94 label detection signal list

95 recording layer

96 address

97 start address

98 end address

99 innermost circumferential track number

100 outermost circumferential track number

111 counter

112 address counter

113 address block counter

114 sub-eject section

115 sub-solution supply section

116 sub-nozzle

118 step

120 mask

121 mask (for DNA spots)

122 hydroxy group

123 A (adenine)

124 C (cytosine)

125 G (guanine)

126 T (thymine)

130 tube

131 probe

132 container

133 sheet

134 mark tube

135 solution

136 mark tube

137 block

138 chip

139 fix plate

140 fix plate ID

141 biomolecule spot

142 mark spot

143 identification mark

144 synchronization mark

145 identification number

146 attribute table

147 test database

148 step (flowchart)

149 test apparatus

150 network

151 memory

152 error correction code

153 mark solution

154 mark biomolecule spot

155 analysis program

156 mark microcapsule

157 synchronization mark

158 synchronization mark

159 original data

160 flat tube

161 rectangular biomolecule spot

162 synchronization mark

170 subject

171 sample

172 biomolecule extraction section

173 specimen

174 main test system

175 test section

176 communication section

177 the Internet

178 sub-test system

179 communication section

180 analysis system

181 analysis section

182 selection section

183 output section

184 (biomolecule spot identification number) attribute database

185 selective output

186 request output

187 diagnosis system

188 diagnosis section

189 treatment policy production section

190 treatment policy output section

191 chip ID-subject correspondence database

192 diagnosis result output section

193 test system

194 black box section

195 input/output section

197 cipher decoding section

198 IC chip

199 electrode

200 substrate

201 non-volatile memory

300 biomolecule chip

301 biomolecule spot

302 equal interval

303 non-equal interval

310 biomolecule chip

311 first biomolecule spot

312 second biomolecule spot

BEST MODE FOR CARRYING OUT THE INVENTION

It should be understood throughout the present specification thatarticles for singular forms (e.g., “a”, “an”, “the”, etc. in English;“ein”, “der”, “das”, “die”, etc. and their inflections in German; “un”,“une”, “le”, “la”, etc. in French; “un”, “una”, “el”, “la”, etc. inSpanish; and articles, adjectives, etc. in other languages) include theconcept of their plurality unless otherwise mentioned. It should be alsounderstood that terms as used herein have definitions ordinarily used inthe art unless otherwise mentioned.

Hereinafter, the meanings of terms as particularly used herein will bedescribed.

The terms “substrate” and “support” as used herein have the samemeaning, i.e., a material for an array construction of the presentinvention (preferably, in a solid form). Examples of a material for thesubstrate include any solid material having a property of binding to abiomolecule used in the present invention either by covalent bond ornoncovalent bond, or which can be derived in such a manner as to havesuch a property.

Such a material for the substrate may be any material capable of forminga solid surface, for example, including, but being not limited to,glass, silica, silicon, ceramics, silica dioxide, plastics, metals(including alloys), naturally-occurring and synthetic polymer (e.g.,polystyrene, cellulose, chitosan, dextran, and nylon). The substrate maybe formed of a plurality of layers made of different materials. Forexample, an inorganic insulating material, such as glass, silica glass,alumina, sapphire, forsterite, silicon carbide, silicon oxide, siliconnitride, or the like, can be used. Moreover, an organic material, suchas polyethylene, ethylene, polypropylene, polyisobutylene, polyethyleneterephthalate, unsaturated polyester, fluorine-containing resin,polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate,polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile,polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urearesin, epoxy resin, melamine resin, styrene acrylonitrile copolymer,acrylonitrilebutadienestyrene copolymer, silicone resin, polyphenyleneoxide, or polysulfone, can be used. In the present invention, a filmused for nucleic acid blotting, such as a nitrocellulose film, a PVDFfilm, or the like, can also be used.

In one embodiment of the present invention, an electrode material can beused for a substrate electrode which serves as both a substrate and anelectrode. In the case of such a substrate electrode, a surface of thesubstrate electrode is separated into electrode regions by an insulatinglayer region. Preferably, different biomolecules are fixed to therespective isolated electrode regions. The electrode material is notparticularly limited. Examples of the electrode material include a metalalone, such as gold, gold alloy, silver, platinum, mercury, nickel,palladium, silicon, germanium, gallium, tungsten, and the like, andalloys thereof, or carbon, such as graphite, glassy carbon, and thelike, or oxides or compounds thereof. Further, a semiconductor compound,such as silicon oxide and the like, or various semiconductor devices,such as CCD, FET, CMOS, and the like, can be used. When a substrateelectrode in which an electrode film is formed on an insulatingsubstrate so that the substrate is integrated with the electrode, theelectrode film can be produced by plating, printing, sputtering,deposition or the like. In the case of deposition, an electrode film canbe formed by a resistance heating method, a high-frequency heatingmethod, an electron-beam heating method, or the like. In the case ofsputtering, an electrode film can be produced by direct currentsputtering, bias sputtering, asymmetric AC sputtering, gettersputtering, high-frequency sputtering, or the like. Furthermore,electropolymerized film, such as polypyrrole, polyaniline, and the like,or a conductive polymer can be used. An insulating material used forseparating the electrode surface in the present invention is notparticularly limited, but is preferably a photopolymer or a photoresistmaterial. Examples of the resist material include a photoresist forlight exposure, a photoresist for ultraviolet radiation, a photoresistfor X ray, and a photoresist for electron beam. Examples of aphotoresist for light exposure include photoresists including cyclizedrubber, polycinnamic acid, and novolac resin as major ingredients. As aphotoresist for ultraviolet radiation, cyclized rubber, phenol resin,polymethylisopropenylketone (PMIPK), polymethylmethacrylate (PMMA), orthe like is used. As a photoresist for X ray, COP, methacrylate, or thelike can be used. As a photoresist for electron beam, theabove-described substances, such as PMMA or the like, can be used.

“Chip” as used herein refers to an ultramicro-integrated circuit havingvarious functions, which constitutes a part of a system. “Biomoleculechip” as used herein refers to a chip comprising a substrate and abiomolecule, in which at least one biomolecule as set forth herein isdisposed on the substrate.

The term “address” as used herein refers to a unique position on asubstrate which can be distinguished from other unique positions. Anaddress is suitably used to access to a biomolecule associated with theaddress. Any entity present at each address can have an arbitrary shapewhich allows the entity to be distinguished from entities present atother addresses (e.g., in an optical manner). The shape of an addressmay be, for example, a circle, an ellipse, a square, or a rectangle, oralternatively an irregular shape.

The size of each address varies depending on, particularly, the size ofa substrate, the number of addresses on the specific substrate, theamount of samples to be analyzed and/or an available reagent, the sizeof a biomolecule, and the magnitude of a resolution required for anymethod in which the array is used. The size of an address may range from1-2 nm to several centimeters (e.g., 1-2 mm to several centimeters,etc., 125×80 mm, 10×10 mm, etc.). Any size of an address is possible aslong as it matches the array to which it is applied. In such a case, asubstrate material is formed into a size and a shape suitable for aspecific production process and application of an array. For example, inthe case of analysis where a large amount of samples to be measured areavailable, an array may be more economically constructed on a relativelylarge substrate (e.g., 1 cm×1 cm or more). Here, a detection systemwhich does not require sensitivity much and is therefore economical maybe further advantageously used. On the other hand, when the amount of anavailable sample to be analyzed and/or reagent is limited, an array maybe designed so that consumption of the sample and reagent is minimized.

The spatial arrangement and forms of addresses are designed in such amanner as to match a specific application in which the microarray isused. Addresses may be densely loaded, widely distributed, or dividedinto subgroups in a pattern suitable for a specific type of sample to beanalyzed. “Array” as used herein refers to a pattern of solid substancesfixed on a solid phase surface or a film, or a group of molecules havingsuch a pattern. Typically, an array comprises biomolecules (e.g., DNA,RNA, protein-RNA fusion molecules, proteins, low-weight organicmolecules, etc.) conjugated to nucleic acid sequences fixed on a solidphase surface or a film as if the biomolecule captured the nucleicsequence. “Spots” of biomolecules may be arranged on an array. “Spot” asused herein refers to a predetermined set of biomolecules.

Any number of addresses may be arranged on a substrate, typically up to10⁸ addresses, in other embodiments up to 10⁷ addresses, up to 10⁶addresses, up to 10⁵ addresses, up to 10⁴ addresses, up to 10³addresses, or up to 10² addresses. Therefore, when one biomolecule isplaced on one address, up to 10⁸ biomolecules can be placed on asubstrate, and in other embodiment up to 10⁷ biomolecules, up to 10⁶biomolecules, up to 10⁵ biomolecules, up to 10⁴ biomolecules, up to 10³biomolecules, or up to 10² biomolecules can be placed on a substrate. Inthese cases, a smaller size of substrate and a smaller size of addressare suitable. In particular, the size of an address may be as small asthe size of a single biomolecule (i.e., this size may be of the order of1-2 nm). In some cases, the minimum area of a substrate is determinedbased on the number of addresses on the substrate.

The term “biomolecule” as used herein refers to a molecule related to anorganism. An “organism” as used herein refers to a biological organicbody, including, but being limited to, an animal, a plant, a fungus, avirus, and the like. A biomolecule includes a molecule extracted from anorganism, but is not so limited. A biomolecule is any molecule capableof having an influence on an organism. Therefore, a biomolecule alsoincludes a molecule synthesized by combinatorial chemistry, and a lowweight molecule capable of being used as a medicament (e.g., a lowmolecular weight ligand, etc.) as long as they are intended to have aninfluence on an organism. Examples of such a biomolecule include, butare not limited to, proteins, polypeptides, oligopeptides, peptides,polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g.,including DNA (such as cDNA and genomic DNA) and RNA (such as mRNA)),polysaccharides, oligosaccharides, lipids, low weight molecules (e.g.,hormones, ligands, signal transduction substances, low-weight organicmolecules, etc.), and complex molecules thereof, and the like. Abiomolecule also includes a cell itself, and a part or the whole oftissue, and the like as long as they can be coupled to a substrate ofthe present invention. Preferably, a biomolecule includes a nucleic acidor a protein. In a preferable embodiment, a biomolecule is a nucleicacid (e.g., genomic DNA or cDNA, or DNA synthesized by PCR or the like).In another preferable embodiment, a biomolecule may be a protein.Preferably, one type of biomolecule may be provided for each address ona substrate of the present invention. In another embodiment, a samplecontaining two or more types of biomolecules may be provided for eachaddress.

The term “protein”, “polypeptide”, “oligopeptide” and “peptide” as usedherein have the same meaning and refer to an amino acid polymer havingany length. This polymer may be a straight, branched or cyclic chain. Anamino acid may be a naturally-occurring or non-naturally-occurring aminoacid, or a variant amino acid. The term may be assembled into a complexof a plurality of polypeptide chains. The term also includes anaturally-occurring or artificially modified amino acid polymer. Suchmodification includes, for example, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification (e.g., conjugation with a labelingcomponent). This definition encompasses a polypeptide containing atleast one amino acid analog (e.g., non-naturally-occurring amino acid,etc.), a peptide-like compound (e.g., peptoid), and other variants knownin the art, for example.

The terms “polynucleotide”, “oligonucleotide”, and “nucleic acid” asused herein have the same meaning and refer to a nucleotide polymerhaving any length. This term also includes an “oligonucleotidederivative” or a “polynucleotide derivative”. An “oligonucleotidederivative” or a “polynucleotide derivative” includes a nucleotidederivative, or refers to an oligonucleotide or a polynucleotide havingdifferent linkages between nucleotides from typical linkages, which areinterchangeably used. Examples of such an oligonucleotide specificallyinclude 2′-O-methyl-ribonucleotide, an oligonucleotide derivative inwhich a phosphodiester bond in an oligonucleotide is converted to aphosphorothioate bond, an oligonucleotide derivative in which aphosphodiester bond in an oligonucleotide is converted to a N3′-P5′phosphoroamidate bond, an oligonucleotide derivative in which a riboseand a phosphodiester bond in an oligonucleotide are converted to apeptide-nucleic acid bond, an oligonucleotide derivative in which uracilin an oligonucleotide is substituted with C-5 propynyl uracil, anoligonucleotide derivative in which uracil in an oligonucleotide issubstituted with C-5 thiazole uracil, an oligonucleotide derivative inwhich cytosine in an oligonucleotide is substituted with C-5 propynylcytosine, an oligonucleotide derivative in which cytosine in anoligonucleotide is substituted with phenoxazine-modified cytosine, anoligonucleotide derivative in which ribose in DNA is substituted with2′-O-propyl ribose, and an oligonucleotide derivative in which ribose inan oligonucleotide is substituted with 2′-methoxyethoxy ribose.

“Gene” as used herein refers to a factor defining a genetic trait. Agene is typically arranged in a certain sequence on a chromosome. A genewhich defines the first-order structure of a protein is called astructural gene. A gene which regulates the expression of a structuralgene is called a regulatory gene. A “gene” as used herein may refer to a“polynucleotide”, an “oligonucleotide” and a “nucleic acid”, and/or a“protein”, a “polypeptide”, an “oligopeptide” and a “peptide”. As usedherein, “homology” of a gene refers to the magnitude of identity betweentwo or more gene sequences. Therefore, the greater the homology betweentwo certain genes, the greater the identity or similarity between theirsequences. Whether or not two genes have homology is determined bycomparing their sequences directly or by a hybridization method understringent conditions. When two gene sequences are directly compared witheach other, the genes have representatively at least 50% homology,preferably at least 70% homology, more preferably at least 80%, 90%,95%, 96%, 97%, 98%, or 99% homology with the DNA sequence of the genesare identical.

The term “polysaccharide”, “complex carbohydrate”, “oligosaccharide”,“sugar”, and “carbohydrate” have the same meaning and refer to a polymercompound in which monosaccharides are dehydrocondensed by glycosidebonds. “Simple sugar” or “monosaccharide” refers to a substancerepresented by the general formula C_(n)H_(2n)O_(n), which cannot bedecomposed by hydrolysis to a simpler molecule. CnH_(2n)O_(n) where n=2,3, 4, 5, 6, 7, 8, 9 and 10, represent diose, triose, tetrose, pentose,hexose, heptose, octose, nonose, and decose, respectively.Monosaccharide generally corresponds to an aldehyde or ketone of chainpolyhydric alcohol, the former being called aldose and the latter beingcalled ketose.

A biomolecule of the present invention may be collected from an organismor may be chemically synthesized by a method known to those skilled inthe art. For example, a synthesis method using an automated solid phasepeptide synthesizer is described in the following: Stewart, J. M. et al.(1984). Solid Phase Peptide Synthesis, Pierce Chemical Co.; Grant, G. A.(1992). Synthetic Peptides: A User's Guide, W. H. Freeman; Bodanszky, M.(1993). Principles of Peptide Synthesis, Springer-Verlag; Bodanszky, M.et al. (1994). The Practice of Peptide Synthesis, Springer-Verlag;Fields, G. B. (1997). Phase Peptide Synthesis, Academic Press;Pennington, M. W. et al. (1994). Peptide Synthesis Protocols, HumanaPress; Fields, G. B. (1997). Solid-Phase Peptide Synthesis, AcademicPress. An oligonucleotide may be prepared by automated chemicalsynthesis using any DNA synthesizer commercially available from AppliedBiosystems or the like. A composition and a method for automatedoligonucleotide synthesis are disclosed in, for example, U.S. Pat. No.4,415,732, Caruthers et al. (1983); U.S. Pat. No. 4,500,707,Caruthers(1985); and U.S. Pat. No. 4,668,777, Caruthers et al. (1987).

In one embodiment of the present invention, a library of biomolecules(e.g., low-weight organic molecules, combinatorial chemistry products)may be coupled to a substrate, and a resultant substrate can be used toproduce a microarray for screening of molecules. A compound library usedin the present invention can be prepared or obtained by any meansincluding, but not limited to, a combinatorial chemistry technique, afermentation method, extraction procedures from plants and cells, or thelike. A method for producing a combinatorial library is well known inthe art. See, for example, E. R. Felder, Chimia 1994, 48, 512-541;Gallop et al., J. Med. Chem. 1994, 37, 1233-1251; R. A. Houghten, TrendsGenet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lamet al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3,171-183; Madden et al., Perspectives in Drug Discovery and Design2,269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6382; Brenner etal., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al., J.Med. Chem. 1994, 37, 1385-1401; Lebl et al., Biopolymers 1995, 37177-198; and literature cited therein. These publications are hereinincorporated by reference in their entirety.

“Stringent conditions” as used herein refers to widely used and wellknown conditions in the art concerning hybridization. Such conditionsare, for example, the following: hybridization is conducted in thepresence of 0.7 to 1.0 M NaCl at 65° C., and thereafter, 0.1 to 2-foldconcentration SSC (saline-sodium citrate) solution (1-fold concentrationSSC solution has a composition of 150 mM sodium Chloride, 15 mM sodiumcitrate) is used to wash a filter at 65° C. Hybridization can beconducted in accordance with a method described in an experimentalmanual, such as Molecular Cloning 2nd ed., Current Protocols inMolecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, APractical Approach, Second Edition, Oxford University Press (1995), orthe like.

Comparison in identity between base sequences is herein calculated by asequence analyzing tool, BLAST, using default parameters.

A method, biomolecule chip and apparatus of the present invention may beused in, for example, diagnosis, forensic medicine, drug search(medicament screening) and development, molecular biological analysis(e.g., array-base nucleotide sequence analysis and array-base genesequence analysis), analysis of protein properties and functions,pharmacogenomics, proteomics, environmental assessment, and otherbiological and chemical analysis.

A method, biomolecule chip and apparatus of the present invention may beused in the detection of various genes. A gene to be detected is notparticularly limited. Examples of such a gene to be detected includegenes of viral pathogens (including, but not limited to, hepatitisviruses (type A, B, C, D, E, F, and G), HIV, influenza viruses, herpesviruses, adenovirus, human polyoma virus, human Papilloma virus, humanParvovirus, mumps virus, human rotavirus, Enterovirus, Japaneseencephalitis virus, dengue virus, rubella virus, and HTLV); genes ofbacterial pathogens (including, but not limited to, Staphylococcusaurens, hemolytic streptococcus, virulent Escherichia coli, enteritisvibrio, Helicobacter pylori, Campylobacter, Vibrio cholerae, dysenterybacillus, Salmonella, Yersinia, gunococcus, Listeria monocytogenes,Leptospira, Legionella, Spirochaeta, Mycoplasma pneumoniae, Rickettsia,and Chlamydia), and genes of Entamoeba histolytica, pathogenic fungi,parasites, and fungi.

A method, biomolecule chip and apparatus of the present invention may beused in detection and diagnosis for neoplastic diseases, such ashereditary diseases, retinoblastoma, Wilms' tumor, familial colonicpolyposis, neurofibromatosis, familial breast cancer, xerodermapigmentosum, brain tumor, cancer of the oral cavity, esophageal cancer,stomach cancer, colon cancer, liver cancer, pancreas cancer, lungcancer, thyroid tumor, tumor of the mammary gland, tumor of urinaryorgans, tumor of male organs, tumor of female organs, skin tumor, tumorof bones and soft parts, leukemia, lymphoma, solid tumor, and the like.

The present invention can also be applied to polymorphism analysis, suchas RFLP analysis, SNP (snipp, single nucleotide polymorphism) analysis,or the like, analysis of base sequences, and the like. The presentinvention can also be used for screening of a medicament.

The present invention can be applied to any situation requiring abiomolecule test other than medical applications, such as food testing,quarantine, medicament testing, forensic medicine, agriculture,husbandry, fishery, forestry, and the like. The present invention isalso intended to be used particularly for the purposes of safety offoods (BSE test).

The present invention may be used to obtain biochemical test data.Examples of items of biochemical tests include, but are not limited to,total protein, albumin, thymol reaction, Kunkel's zinc sulfate testing,plasma ammonia, urea nitrogen, creatinine, uric acid, total bilirubin,direct reacting bilirubin, GOT, GPT, cholinesterase, alkalinephosphatase, leucine aminopeptidase, γ-glutamyl transpeptidase,creatinine phosphakinase, lactic dehydrogenase, amylase, sodium,potassium, chloride ion (chlor), total calcium, inorganic phosphor,serum iron, unsaturated iron-binding capability, serum osmotic pressure,total cholesterol, free cholesterol, HDL-cholesterol, triglyceride,phospholipid, free fatty acid, plasma glucose, insulin, BSP retentionratio, ICG disappearance ratio, ICG retention ratio, spinal fluid•totalprotein, spinal fluid•sugar, spinal fluid•chlorine, urine•total protein,urine•glucose, urine•amylase, urine•ureic acid, urine•urea nitrogen,urine•creatinine, urine•calcium, urine•osmotic pressure, urine•inorganicphosphor, urine•sodium, urine•potassium, urine•chlor,N-acetylglucosamimidase in urine, 1-hour creatinine clearance, 24-hourcreatinine clearance, phenolsulfonephthalein, C-reactive protein, andthe like. A method and principle for measuring these test items are wellknown and commonly used in the art.

The present invention can also be used for detection of a gene amplifiedby PCR, SDA, NASBA, or the like, other than a sample directly collectedfrom an organism. In the present invention, a target gene can be labeledin advance with an electrochemically active substance, a fluorescentsubstance (e.g., FITC, rhodamine, acridine, Texas Red, fluorecein,etc.), an enzyme (e.g., alkaline phosphatase, peroxidase, glucoseoxidase, etc.), acolloid particle (e.g., a hapten, alight-emittingsubstance, an antibody, an antigen, gold colloid, etc.), a metal, ametal ion, a metal chelate (e.g., trisbipyridine, trisphenanthroline,hexamine, etc.), or the like.

In the present invention, a sample to be tested or diagnosed is notparticularly limited and includes, for example, blood, serum,leukocytes, urine, stool, semen, saliva, tissue, cultured cells, sputum,and the like.

In one embodiment, a nucleic acid component is extracted from thesesamples in order to test nucleic acid. The extraction is not limited toa particular method. A liquid-liquid extraction method, such asphenol-chloroform method and the like, or a liquid-solid extractionmethod using a carrier can be used. Alternatively, a commerciallyavailable nucleic acid extraction method QIAamp (QIAGEN, Germany) or thelike can be used. Next, a sample containing an extracted nucleic acidcomponent is subjected to a hybridization reaction on a biomolecule chipof the present invention. The reaction is conducted in a buffer solutionhaving an ionic strength of 0.01 to 5 and a pH of 5 to 10. To thissolution may be added dextran sulfate (hybridization acceleratingagent), salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant, or thelike. The extracted nucleic acid component is added to the solution,followed by heat denaturation at 90° C. or more. Insertion of abiomolecule chip can be carried out immediately after denaturation orafter rapid cooling to 0° C. Alternatively, a hybridization reaction canbe conducted by dropping a solution on a substrate. The rate of areaction can be increased by stirring or shaking during the reaction.The temperature of a reaction is in the range of 10° C. to 90° C. Thetime of a reaction is in the range of one minute to about one night.After a hybridization reaction, an electrode is removed and then washed.For washing, a buffer solution having an ionic strength of 0.01 to 5 anda pH of 5 to 10 can be used.

“Microcapsule” as used herein refers to a microparticle enveloping asubstance with a molecular membrane or the like, or its container-likesubstance. A microcapsule usually has a spherical shape and a size ofseveral micrometers to several hundred micrometers. In general, amicrocapsule can be prepared as follows. A water droplet-in-water typeemulsion is produced, and a polymer thin film is produced by interfacialpolycondensation at an interface between the micro-emulsion particle anda medium so that the particle is covered with the thin film. The capsuleis isolated from the oil by centrifugation, followed by dialysis forpurification. When an emulsion is prepared, an intended biomolecule isdissolved and dispersed into a water phase, so that the biomolecule canbe enveloped in a capsule. The thickness of the thin film is 10 to 20μm. The thin film can be provided with semipermeability or surfacecharge. In the present invention, a microcapsule protects and isolates acontent, such as a biomolecule. Such a content can be optionallydissolved, mixed or allowed to react. In a method for producing abiomolecule substrate according to the present invention, a microcapsuleis sprayed onto a substrate by an ink jet method (e.g., Bubble Jet®,etc.), a PIN method, or the like. The sprayed microcapsule is heated toa temperature higher than the melting point of its shell so that acontent, such as a biomolecule, can be immobilized on the substrate. Inthis case, the substrate is preferably coated with a substance having anaffinity for the biomolecule.

“Label” and “mark” as used herein have the same meaning and refer to anentity which distinguishes an intended molecule or substance from othersubstances (e.g., a substance, energy, electromagnetic wave, etc.).Examples of such a labeling method include an RI (radioisotope) method,a fluorescence method, a biotin method, a chemiluminescence method, andthe like. When both a nucleic acid fragment and its complementaryoligonucleotide are labeled by a fluorescence method, they are labeledwith fluorescence substances having different maximum wavelengths offluoresence. The difference in the maximum wavelength of fluorescence ispreferably at least 10 nm. Any fluorescence substance which can bind toa base portion of nucleic acid can be used. Preferable fluorescencesubstances include cyanine dye (e.g., Cy3, Cy5, etc. in Cy Dye™ series),a rhodamine 6G reagent, N-acetoxy-N-2-acetylaminofluorene (AAF), AAIF(an iodine derivative of AAF), and the like. Examples of a combinationof fluorescence substances having a difference in the maximum wavelengthof fluorescence of at least 10 nm, include a combination of Cy5 and arhodamine 6G reagent, a combination of Cy3 and fluorescein, acombination of a rhodamine 6G reagent and fluorescein, and the like.

“Chip attribute data” as used herein refers to data associated with someinformation relating to a biomolecule chip of the present invention.Chip attribute data includes information associated with a biomoleculechip, such as a chip ID, substrate data, and biomolecule attribute data.“Chip ID” as used herein refers to a code for identification of eachchip. “Substrate data” or “substrate attribute data” as used hereinrefers to data relating to a substrate used in a biomolecule chip of thepresent invention. Substrate data may contain information relating to anarrangement or pattern of a biomolecule. “Biomolecule attribute data”refers to information relating to a biomolecule, including, for example,the gene sequence of the biomolecule (a nucleotide sequence in the caseof nucleic acid, and an amino acid sequence in the case of protein),information relating to a gene sequence (e.g., a relationship betweenthe gene and a specific disease or condition), a function in the case ofa low weight molecule or a hormone, library information in the case of acombinatorial library, molecular information relating to affinity for alow weight molecule, and the like. “Personal information data” as usedherein refers to data associated with information for identifying anorganism or subject to be measured by a method, chip or apparatus of thepresent invention. When the organism or subject is a human, personalinformation data includes, but is not limited to, age, sex, healthcondition, medical history (e.g., drug history), educational background,the company of your insurance, personal genome information, address,name, and the like. When personal information data is of a domesticanimal, the information may include data about the production company ofthe animal. “Measurement data” as used herein refers to raw data as aresult of measurement by a biomolecule substrate, apparatus and systemof the present invention and specific processed data derived therefrom.Such raw data may be represented by the intensity of an electric signal.Such processed data may be specific biochemical data, such as a bloodsugar level and a gene expression level.

“Recording region” as used herein refers to a region in which data maybe recorded. In a recording region, measurement data as well as theabove-described chip attribute data can be recorded.

In a preferable embodiment of the present invention, personalinformation data and biomolecule attribute data or measurement data maybe separately managed. By managing these data separately, the secrecy ofhealth-related information, i.e., personal privacy, can be protected.Moreover, in the case of medicament screening, even if screening isfarmed out to an outside company, data can be obtained without leakingto secret information to the outside company. Therefore, the presentinvention can be applied to outsourcing in which secret information isprotected.

(General Techniques)

Techniques as used herein are well known techniques commonly used inmicrofluidics, micromachining, organic chemistry, biochemistry, geneticengineering, molecular biology, genetics, and their related fields within the technical scope of the art, unless otherwise specified. Thesetechniques are sufficiently described in, for example, literature listedbelow and described elsewhere herein.

Micromachining is described in, for example, Campbell, S. A. (1996). TheScience and Engineering of Microelectronic Fabrication, OxfordUniversity Press; Zaut, P. V. (1996). Micromicroarray Fabrication: aPractical Guide to Semiconductor Processing, Semiconductor Services;Madou, M. J. (1997). Fundamentals of Microfabrication, CRC1 5 Press;Rai-Choudhury, P. (1997). Handbook of Microlithography, Micromachining,& Microfabrication: Microlithography; and the like, related portions ofwhich are herein incorporated by reference.

Molecular biology and recombinant DNA techniques are described in, forexample, Maniatis, T. et al. (1982). Molecular Cloning: A LaboratoryManual, Cold SpringHarbor; Ausubel, F. M. (1987). Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-Interscience;Ausubel, F. M. (1989). Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Sambrook, J. et al.(1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor;Innis, M. A. (1990). PCR Protocols: A Guide to Methods and Applications,Academic Press; Ausubel, F. M. (1992). Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Greene Pub. Associates; Innis, M. A. et al. (1995).PCR Strategies, Academic Press; Ausubel, F. M. (1999). Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Wiley, and annual updates; Sninsky, J. J. et al.(1999). PCR Applications: Protocols for Functional Genomics, AcademicPress; and the like, related portions of which are herein incorporatedby reference.

Nucleic acid chemistry, such as DNA synthesis techniques and the like,is described in, for example, Gait, M. J. (1985). OligonucleotideSynthesis: A Practical Approach, IRL Press; Gait, M. J. (1990).Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.(1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press;Adams, R. L. et al. (1992). The Biochemistry of the Nucleic Acids,Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistryof Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). NucleicAcids in Chemistry and Biology, Oxford University Press; Hermanson, G.T. (I 996). Bioconjugate Techniques, Academic Press; and the like,related portions of which are herein incorporated by reference.

Photolithography is a technique developed by Fodor et al., in which aphotoreactive protecting group is utilized (see Science, 251,767(1991)). A protecting group for a base inhibits a base monomer of thesame or different type from binding to that base. Thus, a base terminusto which a protecting group is bound has no new base-binding reaction. Aprotecting group can be easily removed by irradiation. Initially, aminogroups having a protecting group are immobilized throughout a substrate.Thereafter, only spots to which a desired base is to be bound areselectively irradiated by a method similar to a photolithographytechnique usually used in a semiconductor process, so that another basecan be introduced by subsequent binding into only the bases in theirradiated portion. Now, desired bases having the same protecting groupat a terminus thereof are bound to such bases. Thereafter, the patternof a photomask is changed, and other spots are selectively irradiated.Thereafter, bases having a protecting group are similarly bound to thespots. This process is repeated until a desired base sequence isobtained in each spot, thereby preparing a DNA array. Photolithographytechniques may be herein used.

An ink jet method (technique) is a technique of projecting considerablysmall droplets onto a predetermined position on a two-dimensional planeusing heat or a piezoelectric effect. This technique is widely usedmainly in printers. In production of a DNA array, an ink jet apparatusis used, which has a configuration in which a piezoelectric device iscombined with a glass capillary. A voltage is applied to thepiezoelectric device which is connected to a liquid chamber, so that thevolume of the piezoelectric device is changed and the liquid within thechamber is expelled as a droplet from the capillary connected to thechamber. The size of the expelled droplet is determined by the diameterof the capillary, the volume variation of the piezoelectric device, andthe physical property of the liquid. The diameter of the droplet isgenerally 30 μm. An ink jet apparatus using such a piezoelectric devicecan expel droplets at a frequency of about 10 KHz. In a DNA arrayfabricating apparatus using such an ink jet apparatus, the ink jetapparatus and a DNA array substrate are relatively moved so thatdroplets can be dropped onto desired spots on the DNA array. DNA arrayfabricating apparatuses using an ink jet apparatus are roughly dividedinto two categories. One category includes a DNA array fabricatingapparatus using a single ink jet apparatus, and the other includes a DNAarray fabricating apparatus using a multi-head ink jet apparatus. TheDNA array fabricating apparatus with a single ink jet apparatus has aconfiguration in which a reagent for removing a protecting group at aterminus of an oligomer is dropped onto desired spots. A protectinggroup is removed from a spot, to which a desired base is to beintroduced, by using the ink jet apparatus so that the spot isactivated. Thereafter, the desired base is subjected to a bindingreaction throughout a DNA array. In this case, the desired base is boundto only spots having an oligomer whose terminus is activated by thereagent dropped from the ink jet apparatus. Thereafter, the terminus ofa newly added base is protected. Thereafter, a spot from which aprotecting group is removed is changed and the procedures are repeateduntil desired nucleotide sequences are obtained. On the other hand, in aDNA array fabricating apparatus using a multi-head ink jet apparatus, anink jet apparatus is provided for each reagent containing a differentbase, so that a desired base can be bound directly to each spot. A DNAarray fabricating apparatus using a multi-head ink jet apparatus canhave a higher throughput than that of a DNA array fabricating apparatususing a single ink jet apparatus. Among methods for fixing apresynthesized oligonucleotide to a substrate is a mechanicalmicrospotting technique in which liquid containing an oligonucleotide,which is attached to the tip of a stainless pin, is mechanically pressedagainst a substrate so that the oligonucleotide is immobilized on thesubstrate. The size of a spot obtained by this method is 50 to 300 μm.After microspotting, subsequent processes, such as immobilization usingUV light, are carried out.

BEST MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention provides a method for fabricating abiomolecule substrate. This method comprises the steps of: 1) providinga set of biomolecules and a substrate; 2) enclosing the set ofbiomolecules into microcapsules on thebiomolecule-type-by-biomolecule-type basis; and 3) spraying thebiomolecule microcapsules onto the substrate. Preferably, the set ofbiomolecules are uniform. In a preferred embodiment, the method providesa plurality of sets of biomolecules. Preferably, the microcapsules ofthe set of biomolecules of different types are disposed at differentpositions. In one embodiment, the present invention may further comprisethe step of washing the biomolecule microcapsules after the enclosingstep.

The spraying step used in the method of the present invention isperformed by an ink jet method (including a BUBBLE JET® ink jet method),a PIN method, or the like. Preferably, the spraying step may beperformed by a BUBBLE JET® ink jet method. This is because themicrocapsules may be efficiently immobilized.

In a preferred embodiment, the method may further comprise the step ofsetting the temperature of a solution used in the spraying step to behigher than the melting point of a shell of the biomoleculemicrocapsulate. Such an increased temperature of the solution can leadto efficient immobilization of the biomolecules.

In this biomolecule substrate fabrication method, the biomolecule may bea naturally-occurring or synthetic biomolecule. Examples of such abiomolecule include, but are not limited to, a protein, a polypeptide,an oligopeptide, a peptide, apolynucleotide, an oligonucleotide, anucleotide, nucleic acid (e.g., including DNA, such as cDNA or genomicDNA, and RNA, such as mRNA), a polysaccharide, an oligosaccharide,lipid, a low weight molecule (e.g., a hormone, a ligand, a signaltransduction substance, a low-weight organic molecule, etc.), andcomposite molecules thereof.

Preferably, the biomolecule substrate fabrication method of the presentinvention may further comprise the step of perform labeling specific toeach microcapsule.

In another aspect, the present invention provides a biomolecule chip.This biomolecule chip comprises: a substrate; and biomolecules and chipattribute data arranged on the substrate. The chip attribute data isarranged in the same region as that of the biomolecules. By placing thebiomolecules and the chip attribute data in the same region, anefficient testing can be perfomred.

In one embodiment, the above-described chip attribute data may containinformation relating to chip ID and the substrate. In anotherembodiment, the biomolecule chip of the present invention may furthercomprise a recording region, wherein the recording region is placed onthe same substrate as that of the biomolecule and the chip attributedata, and at least one of the subject data and measurement data isrecorded in the recording region. Preferably, both the subject data andmeasurement data may be recorded in the above-described recordingregion. Note that when it is intended to protect privacy depending onthe purpose, only a part of these pieces of information may be recordedin the recording region. In this case, such data may be encrypted andthen recorded.

Preferably, the above-described chip attribute data may be recorded insuch a manner that the data can be read out by the same means as thatfor detecting the above-described biomolecule. Examples of suchdetection means include, but are not limited to, any means capable ofdetecting the biomolecule, such as a fluorescence analysis apparatus, aspectrophotometer, a scintillation counter, and a luminometer. Sinceboth the chip attribute data and the biomolecule can be read out by thesame detection means, both testing of raw data and reading ofmeasurement conditions can be performed by a single read-out operation,thereby making it possible to significantly reduce an operation time andsimplifying signal sending and receiving equipment.

In a preferred embodiment, a specific mark may be attached to theabove-described substrate. By attaching the specific mark to thesubstrate, identification of the substrate can be double-checked,thereby making it possible to reduce diagnosis and testing errors. Inanother preferred embodiment, the specific mark is arranged based on thechip attribute data. By providing such a specific mark, it is possibleto easily read out chip attribute data.

In another embodiment, the above-described chip attribute data maycontain the above-described biomolecule attribute data. By adding thebiomolecule attribute data to the biomolecule chip, various tests anddiagnoses can be performed by using only the chip. In anotherembodiment, this chip attribute data can be maintained in another site.By maintaining the data in another site, personal information can beprevented from being unintentionally leaked even when the biomoleculechip is unintentionally passed to a third party.

In another embodiment, information relating to an address of theabove-described biomolecule may be further recorded. Examples of suchaddress information include geometric information of an arrangement or apattern defined in the present invention. By adding address-relatedinformation to the biomolecule chip, a stand-alone test can beperformed. The address-related information can also be maintained inanother site. By maintaining the information in another site, personalinformation can be prevented from being unintentionally leaked even whenthe biomolecule chip is unintentionally passed to a third party. In apreferred embodiment, the address may be a tracking address.

In a further preferred embodiment, the above-described chip attributedata may be encrypted. The whole or a part of the data may be encrypted.Preferably, personal information data, biomolecule attribute data, andmeasurement data may be encrypted. These data may be encrypted byseparate encryption means. Such an encryption means is well known in theart, including, for example, a means using a public key. The presentinvention is not so limited.

In another embodiment, data relating to a label used to detect thebiomolecule may be recorded. Examples of such a label include, but arenot limited to, any substance for labeling a biomolecule, such as, forexample, a fluorescent molecule, a chemoluminescent molecule, aradioactive isotope, and the like. By providing such label-related data,a test or diagnosis can be performed by using only a biomolecule chip.Preferably, the label-related data contains at least one of thewavelength of excited light and the wavelength of fluorescence, and morepreferably both of them.

The biomolecule used in the biomolecule chip of the present inventionmay be a naturally-occurring or synthetic biomolecule. Examples of sucha biomolecule include, but are not limited to, a protein, a polypeptide,an oligopeptide, a peptide, apolynucleotide, an oligonucleotide, anucleotide, nucleic acid (e.g., including DNA, such as cDNA or genomicDNA, and RNA, such as mRNA), a polysaccharide, an oligosaccharide,lipid, a low weight molecule (e.g., a hormone, a ligand, a signaltransduction substance, a low-weight organic molecule, etc.), andcomposite molecules thereof. Preferably, the biomolecule may be anucleic acid or a protein, and more preferably DNA (e.g., cDNA orgenomic DNA). In another preferred embodiment, the biomolecule may beDNA amplified by an amplification means, such as PCR or the like. Inanother preferred embodiment, the biomolecule may be a synthesizedprotein.

In another aspect, the present invention provides a biomolecule chipcomprising: 1) a substrate; and 2) biomolecules arranged on thesubstrate, wherein spots of the biomolecules are spaced by at least onenon-equal interval, an address of the biomolecule spot can be identifiedfrom the non-equal interval. By providing at least one non-equalinterval, the interval can be used as a reference to identify therelative positions of other spots. With this structure, it is possibleto identify the address of a spot having interaction only by the stepsof detecting all biomolecules and detecting a spot after contacting asample, without a step of identifying the position of the spot. Such anaddress identifying method is also herein called address identificationusing specific “arrangement”. FIG. 47 shows an example of addressspecification using specific arrangement. In FIG. 47, biomolecules arespaced at equal intervals as indicated by 302, except that at least oneinterval between biomolecules is a non-equal interval as indicated by303. When this non-equal interval is used as a starting point, theaddress of any spot can be identified.

Preferably, the non-equal interval is modulated. Modulation as usedherein refers to variations in spot interval. Modification may be eitherregular or irregular. An example of such modulation is a sequence of 00,01, 10, 00, 01, 01, 01 in the binary number system. The presentinvention is not so limited. By changing modulation, more efficientaddress identification can be made possible.

In a certain embodiment, the above-described non-equal intervals may bepresent in at least two directions. Preferably, the non-equal intervalsin the two directions may be distinguished from each other. By using thenon-equal intervals in at least two directions, address can be reliablyidentified even if data is read out in the case when the substrate isturned upside down. Preferably, a plurality of such non-equal intervalsmay be present. Moreover, such non-equal intervals can be scattered on asubstrate.

In another embodiment, the present invention provides a biomolecule chipcomprising: 1) a substrate: and 2) biomolecules arranged on thesubstrate, wherein the biomolecules include a distinguishable firstbiomolecule and a distinguishable second biomolecule, an address of thebiomolecule can be identified based on an arrangement of spots of thefirst biomolecules and spots of the second biomolecule. By providing atleast two types of distinguishable biomolecules, it is possible toidentify the address of a spot having interaction only by the steps ofdetecting all biomolecules and detecting a spot after contacting asample, without a step of identifying the position of the spot. Such anaddress identifying method is also called address identification using aspecific “pattern”. FIG. 48 shows an example of address identificationusing a specific pattern. In FIG. 48, a first biomolecule 311 can bedistinguished from a second biomolecule 312. In this example, by usingthe second biomolecule 312 as a starting point, the address of any spotcan be identified.

“Distinguishable” as used herein indicates that identification can becarried out by at least one detection means (including, not limited to,the naked eye, a fluorescence measurement apparatus, aspectrophotometer, a radiation measurement apparatus, etc.). Therefore,a distinguishable biomolecule may be, for example, a molecule which canbe identified by the naked eye, or a molecule which emits differentfluorescence when it is excited. “Distinguishable” also indicates thatidentification can be carried out by the same label having a differentlevel (e.g., a difference in the amount of dye, etc.).

In one embodiment of the biomolecule chip of the present invention inwhich an address is identified by a specific arrangement or a specificpattern, a label distinguishable from the biomolecule may be placedbetween the biomolecule spots. Such a label may be any label as definedherein, and preferably a label which can be detected by the samedetection means as the above-described means for detecting abiomolecule.

In one embodiment of the biomolecule chip of the present invention inwhich an address is identified by a specific arrangement or a specificpattern, the above-described distinguishable label can be detected by adetection means. Examples of such detection means include, but are notlimited to, any means capable of detecting the biomolecule, such as afluorescence analysis apparatus, a spectrophotometer, a scintillationcounter, and a luminometer.

In one embodiment of the biomolecule chip of the present invention inwhich an address is identified by a specific arrangement or a specificpattern, the label may be arranged in a horizontal direction and avertical direction on the substrate.

In one embodiment of the biomolecule chip of the present invention inwhich an address is identified by a specific arrangement or a specificpattern, a synchronization mark may be arranged. By providing asynchronization mark, address identification is made easier.

A biomolecule used in one embodiment of the biomolecule chip of thepresent invention in which an address is identified by a specificarrangement or a specific pattern may be a naturally-occurring orsynthetic biomolecule. Examples of such a biomolecule include, but arenot limited to, a protein, a polypeptide, an oligopeptide, a peptide, apolynucleotide, an oligonucleotide, a nucleotide, nucleic acid (e.g.,including DNA, such as cDNA or genomic DNA, and RNA, such as mRNA), apolysaccharide, an oligosaccharide, lipid, a low weight molecule (e.g.,a hormone, a ligand, a signal transduction substance, a low-weightorganic molecule, etc.), and composite molecules thereof. Preferably,the biomolecule may be a nucleic acid or a protein, and more preferablyDNA (e.g., cDNA or genomic DNA). In another preferred embodiment, thebiomolecule may be DNA amplified by an amplification means, such as PCRor the like.

In another aspect, the present invention provides a biomolecule chip.This biomolecule chip comprises: 1) a substrate; and 2) biomoleculesarranged on the substrate, wherein spots storing attribute data arearranged on a side of the substrate opposite to a side on which spots ofthe biomolecules are arranged. By arranging the spots storing attributedata on the rear side of the biomolecule chip, both data can be detectedby a single read-out operation so that testing and/or diagnosis can beperformed. Preferably, this attribute data may contain addressinformation. The attribute data may contain biomolecule attribute dataand the like.

In another aspect, the present invention provides a biomolecule chipcomprising: 1) a substrate; 2) biomolecules arranged on the substrate;and 3) a data recording region. By providing such a data recordingregion, it is possible to perform testing and/or diagnosis using only abiomolecule chip. Preferably, the data recording region may be placed ona side of the substrate opposite to a side on which spots of thebiomolecules are arranged.

In one aspect, the present invention provides a method for detecting alabel of a biomolecule chip. This method comprises the steps of: 1)providing a biomolecule chip on which at least one labeled biomoleculeis arranged; 2) switching detection elements sequentially for detectingthe biomolecules on the biomolecule chip; and 3) identifying a signaldetected by the detection element. With this method, a signal can bedetected efficiently and in real time in a biomolecule chip. Preferably,this method further comprise: 4) adding up each detected signal. In oneembodiment, this signal may be separated using a wavelength separationmirror. In another embodiment, the above-described biomolecule substratemay further comprise a synchronization mark, and the label may beidentified based on the synchronization mark. By providing thesynchronization mark, an address can be smoothly identified. In anotherembodiment, the biomolecule substrate contains address information on arear side of the biomolecule, and the label is identified based on theaddress information.

In another aspect, the present invention provides a method for testinginformation from an organism. This method comprises the steps of: 1)providing a biomolecule sample from the organism; 2) providing abiomolecule chip of the present invention; 3) contacting the biomoleculesample to the biomolecule chip, and placing the biomolecule chip underconditions which causes an interaction between the biomolecule sampleand a biomolecule placed on the biomolecule; and 4) detecting a signalcaused by the biomolecule and a signal caused by the interaction,wherein the signal is an indicator for at least one informationparameter of the organism, and the signal is related to an addressassigned to the non-equal interval or the spot arrangement.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the sample contains a protein andthe biomolecule placed on the biomolecule chip is an antibody, or thesample contains an antibody and the biomolecule placed on thebiomolecule chip is a protein. In this detection method, hybridizationbetween nucleic acids is detected. This hybridization may be performedunder various stringency conditions. When SNP is detected, stringenthybridization conditions maybe used. When a gene having a relationshipbut being far with respect to species is searched for, moderatehybridization conditions may be used. Such hybridization conditions canbe determined by those skilled in the art from the well-known routinetechniques, depending on the situation.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the sample contains a protein andthe biomolecule placed on the biomolecule chip is an antibody, or thesample contains an antibody and the biomolecule placed on thebiomolecule chip is a protein. In this detection method, anantigen-antibody reaction is detected. An antigen-antibody reaction maybe detected under various stringency conditions. The antibody may beeither a monoclonal antibody or polyclonal antibody. Preferably, theantibody may be a monoclonal antibody. The antibody may be a chimeraantibody, a humanized antibody, or the like.

In a preferred embodiment, the method of the present invention furthercomprises labeling the biomolecule sample with a label molecule. Bylabeling a sample with a desired label molecule, a desired detectionmeans can be used.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the label molecule may bedistinguished from the biomolecule placed on the biomolecule chip. Byproviding a label which can be distinguished from a biomolecule, it iseasy to detect a spot in which an interaction occurs. The label whichcan be distinguished from a biomolecule refers to a label which can bedistinguished from a biomolecule by at least one detection means asdescribed above.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the above-described label moleculecontains a fluorescent molecule, a photophorescent molecule, achemoluminescent molecule, or a radioactive isotope. In this case, adetection means corresponding to the type of label molecule may be used.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the signal detecting step may beperformed either at a site different from where the interaction occursor at the same site as where the interaction occurs. When the signaldetecting step is performed at a different site, the signal may beencrypted. Such encryption is well known in the art. For example,encryption using a public key may be used. By performing detection at adifferent site, it maybe possible to outsource diagnosis or testing.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the method may further comprisesubjecting the signal to filtering so as to extract only signalsrelating to required information. This step may be required forprotecting personal information when outsourcing testing.

In another aspect, the present invention provides a method fordiagnosing a subject. The method comprises the steps of: 1) providing asample from the subject; 2) providing a biomolecule chip of the presentinvention; 3) contacting the biomolecule sample to the biomolecule chip,and placing the biomolecule chip under conditions which causes aninteraction between the biomolecule sample and a biomolecule placed onthe biomolecule; 4) detecting a signal caused by the biomolecule and asignal caused by the interaction, wherein the signal is at least onediagnostic indicator for the subject, and the signal is related to anaddress assigned to the non-equal interval or the spot arrangement; and5) determining the diagnostic indicator from the signal.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the sample is nucleic acid, and thebiomolecule placed on the biomolecule chip is nucleic acid. In thisdetection method, hybridization between nucleic acids is detected. Thishybridization may be performed under various stringency conditions. WhenSNP is detected, stringent hybridization conditions may be used. Byplacing nucleic acid relating to a specific disease on a biomoleculechip, a singnal caused by hybridization may be an indicator for thespecific disease.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the sample contains a protein andthe biomolecule placed on the biomolecule chip is an antibody, or thesample contains an antibody and the biomolecule placed on thebiomolecule chip is a protein. In this test method, an antigen-antibodyreaction is detected. The antigen-antibody reaction may be detectedunder various stringency conditions. By placing a protein or an antibodyrelating to a specific disease or condition on a biomolecule chip, adetected signal may be an indicator relating to the specific disease orcondition.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the method further compriseslabeling the sample with a label molecule. By labeling a sample with adesired label, a desired detection means can be used. The label moleculemaybe distinguishable from a biomolecule placed on the above-describedbiomolecule chip. By providing a label which can be distinguished from abiomolecule, it is easy to detect a spot having an interaction.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the above-described label moleculemay contain a fluorescent molecule, a phosphorescent molecule, achemoluminescent molecule, or a radioactive isotope. In this case, adetection means corresponding to the type of the label molecule may beused.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the diagnostic indicator may be anindicator for a disease or a disorder. In another embodiment, thediagnostic indicator may be based on single nucleotide polymorphism(SNP). This diagnostic indicator may be related to a genetic disease. Inanother embodiment, the diagnostic indicator may be based on theexpression level of a protein. The diagnostic indicator may be based ona test result of a biochemical test. A plurality of test values based onthe biochemical tests may be used.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the determining step may beperformed either at a site different from where the interaction occursor at the same site as where the interaction occurs. When thedetermining step is performed at a different site, the present inventionmay further comprise encrypting the signal. By performing detection at adifferent site, it maybe possible to outsource diagnosis or testing.Such outsourcing corresponds to industrially applicable work.

In a preferred embodiment of the method of the present invention fortesting information on an organism, the method may further comprisesubjecting the signal to filtering so as to extract only signalsrelating to required information. This step may be required for avoidingexcessive leakage of personal information to protect the personalinformation when outsourcing testing.

In a preferred embodiment of the method of the present invention fortesting information on an organism, in the detecting step biomoleculeattribute data is hidden, and in the determining step personalinformation data is hidden. Thereby, the whole information required fordiagnosis is prevented from being concentrated into a person or entity,whereby personal information can be protected.

In another aspect, the present invention provides a test apparatusinformation on an organism. This apparatus comprises: 1) a biomoleculechip of the present invention; 2) a sample applying section in fluidcommunication with the biomolecule chip; 3) a reaction control sectionfor controlling a contact and an interaction between the biomoleculeplaced on the biomolecule and a biomolecule sample applied from thesample applying section; and 4) a detection section for detecting asignal caused by the interaction, wherein the signal is an indicator forat least one information parameter of the organism, and the signal isrelated to an address assigned to the non-equal interval or the spotarrangement. This apparatus can perform testing of biologicalinformation without additional address identification.

In a preferred embodiment, the test apparatus of the present inventionfurther comprises a section for receiving and sending the signal. Byproviding the section for receiving and sending the signal, it ispossible to send or receive information to or from the outside. Thissending and receiving section may be connected to a recording apparatusdrive, such as a flexible disk drive, an MO drive, a CD-R drive, a DVD-Rdrive, or a DVD-RAM drive; or a network, such as the Internet or anintranet.

In a preferred embodiment, the test apparatus of the present inventionfurther comprises a region for recording the signal. By providing therecording region, it is possible to store a result of a test. When thetest apparatus is used a plurality of times, stored test results can becompared with each other.

In another aspect, the present invention provides a diagnosis apparatusfor a subject. The apparatus comprises: 1) a biomolecule chip of thepresent invention; 2) a sample applying section in fluid communicationwith the biomolecule chip; 3) a reaction control section for controllinga contact and an interaction between the biomolecule placed on thebiomolecule and a biomolecule sample applied from the sample applyingsection; 4) a detection section for detecting a signal caused by thebiomolecule and a signal caused by the interaction, wherein the signalis an indicator for at least one information parameter of the organism,and the signal is related to an address assigned to the non-equalinterval or the spot arrangement; and 5) determining the diagnosticindicator from the signal. This apparatus can perform testing of subjectinformation without additional address identification.

In a preferred embodiment, the test apparatus of the present inventionfurther comprises a section for receiving and sending the signal. Byproviding the section for receiving and sending the signal, it ispossible to send or receive information to or from the outside. Thissending and receiving section may be connected to a recording apparatusdrive, such as a flexible disk drive, an MO drive, a CD-R drive, a DVD-Rdrive, or a DVD-RAM drive; or a network, such as the Internet or anintranet.

In a preferred embodiment, the test apparatus of the present inventionfurther comprises a region for recording the signal. By providing therecording region, it is possible to store a result of diagnosis. Whenthe test apparatus is used a plurality of times, stored diagnosisresults can be compared with each other.

In another aspect, A biological test system, comprising: A) a main subsystem, comprising: 1) a biomolecule chip of the present invention; 2) asample applying section in fluid communication with the biomoleculechip; 3) a reaction control section for controlling a contact and aninteraction between the biomolecule placed on the biomolecule and abiomolecule sample applied from the sample applying section; 4) adetection section for detecting a signal caused by the biomolecule and asignal caused by the interaction, wherein the signal is an indicator forat least one information parameter of the organism, and the signal isrelated to an address assigned to the non-equal interval or the spotarrangement; and 5) a sending and receiving section for sending andreceiving a signal, and B) a sub sub system, comprising: 1) a sendingand receiving section for sending and receiving a signal; and 2) a testsection for calculating a test value from the signal received from themain sub system, wherein the main sub system and the sub sub system areconnected together via a network.

Preferably, the main sub system and the sub sub system are connectedtogether via a network.

In another preferred embodiment, the signal received by the sub subsystem contains a signal relating to measurement data measured by thesub sub system.

More preferably, the attribute data contains chip ID, personalinformation data, and biomolecule attribute data; the main sub systemcontains the chip ID and the personal information data, but does notcontain the biomolecule attribute data; and the sub sub system containsthe chip ID and the biomolecule attribute data, but does not contain thepersonal information data, and the sub sub system sends the test value,determined in response to a request, to the main sub system. Therefore,the biological test system of the present invention prevents leakage ofinformation to a third party. If information is leaked, privacy can beprotected in testing an organism. In a preferred embodiment, the signalto be sent and received is encrypted.

Preferably, the above-described network may be the Internet or othernetworks (e.g., an intranet, etc.).

In another aspect, the present invention provides a diagnosis systemcomprising: A) a main sub system, comprising: 1) the biomolecule chip ofthe present invention; 2) a sample applying section in fluidcommunication with the biomolecule chip; 3) a reaction control sectionfor controlling a contact and an interaction between the biomoleculeplaced on the biomolecule and a biomolecule sample applied from thesample applying section; 4) a detection section for detecting a signalcaused by the biomolecule and a signal caused by the interaction,wherein the signal is an indicator for at least one informationparameter of the organism, and the signal is related to an addressassigned to the non-equal interval or the spot arrangement; and 5) asending and receiving section for sending and receiving a signal, and B)a sub sub system, comprising: 1) a sending and receiving section forsending and receiving a signal; and 2) a determination section fordetermining the diagnostic indicator from the signal received from themain sub system. The main sub system and the sub sub system areconnected together via a network. In a preferred embodiment, the signalto be sent and received is encrypted.

Preferably, the signal received by the sub sub system contains a signalrelating to measurement data measured by the sub sub system. Morepreferably, the attribute data contains chip ID, personal informationdata, and biomolecule attribute data, the main sub system contains thechip ID and the personal information data, but does not contain thebiomolecule attribute data, and the sub sub system contains the chip IDand the biomolecule attribute data, and data for determining adiagnostic indicator from biomolecule attribute data, but does notcontain the personal information data, and the sub sub system sends thediagnostic indicator, determined in response to a request, to the mainsub system. Therefore, the diagnosis system of the present inventionprevents leakage of information to a third party. If information isleaked, privacy can be protected in diagnosis.

Preferably, the above-described network may be the Internet or othernetworks (e.g., an intranet, etc.).

In another aspect, A test apparatus for biological informationcomprising: a support for a substrate; a plurality of groups ofbiomolecules arranged on the substrate, each group containing thebiomolecules of the same type; shifting means for shifting thesubstrate; a light source for exciting a fluorescence substance labelinga sample to be tested; and optical means for converging light from thelight source. The light source is caused to emit light intermittently inresponse to an intermittent emission signal so as to excite thefluorescence substance, fluorescence from the fluorescence substance isdetected by a photodetector during a period of time when theintermittent emission signal is paused, identification information isreproduced from an arrangement of the DNAs, and the biomoleculesemitting fluorescence is identified.

Preferably, the test apparatus further comprises means for adding updetected detection signals. In another preferred embodiment, the testapparatus further comprises a wavelength separation mirror.

In another aspect, the present invention provides use of a biomoleculechip of the present invention for fabricating an apparatus for testingbiological information.

In another aspect, the present invention provides use of a biomoleculechip of the present invention for fabricating an apparatus fordiagnosing a subject.

In still another aspect, the present invention provides use of abiomolecule of the present invention for screening for a medicament andfabricating an apparatus for screening for a medicament. The presentinvention also provides a biomolecule chip for medicament screening. Thepresent invention also provides a screening apparatus for medicamentscreening. The present invention also provides a method for screeningfor a medicament using a biomolecule chip of the present invention.These method, apparatus, and biomolecule chip have a fundamentalstructure constructed by the same principle as that of testing anddiagnosis for a biomolecule, which can be implemented by those skilledin the art with reference to the present specification.

Hereinafter, the present invention will be described by way of examplesillustrating best mode embodiments. Examples described below are onlyfor illustrative purposes. Therefore, the scope of the present inventionis limited only by the scope of the claims, but not to the examples.

EXAMPLES

Hereinafter, best mode embodiments of the present invention will bedescribed by way of examples with reference to FIGS. 1 to 46.

Example 1 Fabrication Example of Biomolecule Chip (1)

In this example, a method for arranging and immobilizing capture DNAs 2having different sequences on a substrate 1 will be described.

FIG. 1( a) is a top view of DNA spots 2 in which a group of DNAfragments having a specific sequence are fixed on the substrate 1 in theshape of a dot according to the present invention. FIG. 1( b) is across-sectional view thereof. The substrate 1 is usually made of glassand may be made of a plastic. The shape of the substrate 1 may be asquare like a DNA chip, or a circle. DNA dots 2 each contain a differentcapture DNA which is immobilized on the substrate 1. The size of the DNAdot is 100 to 200 μm in diameter in the case of a microarray, and 10 to30 μm in the case of a DNA chip.

A method for forming DNA spots will be described with reference to FIGS.2 and 3. As shown in FIG. 2, (1) shows a capture DNA 3. A method ofpreparing capture DNA is omitted. The capture DNA and labeled DNA with asubject label are subjected to hybridization so as to predict thesequence of subject DNA. (2) shows a DNA solution 4 containing thecapture DNA 3. (3) shows a DNA microcapsule 6 in which the DNA solution4 is covered with a covering 5. (4) shows a container 11 in which theDNA microcapsule 6 is dispersed in a solution 8. (5) shows amicrocapsule 9 in which the DNA microcapsules 6 shown in (4) arecollected and enveloped together with the solution 8 with asub-membrane.

This microcapsulation makes it possible to select separately twosolutions, i.e., the main solution 4 of DNA and the sub-solution 8 ofthe DNA microcapsule. As the DNA solution 4, a solution optimal to DNAor a solution required to immobilize the DNA 3 to the substrate 1 can beselected. As the sub-solution 8, a solution having an optimal viscosityor washing attachability when DNA is arranged on the substrate 1 in aPIN method or an ink jet method can be selected.

FIG. 3 shows a method for arranging 1^(st) to K^(th) DNA spots on thesubstrate 1 by a pinning spot method. Initially, on a tray 12, severalhundreds to several thousands of containers 11 (FIG. 2(4)) containingcapture DNA having a different sequence are arranged in the order of DNAnumbers. As shown in FIGS. 4(1), (2), (3) and (4), at (1) a moving pin14 is moved so that the DNA microcapsule can be attached to the movingpin 14 from the DNA container 11; at (2) and (3) the DNA microcapsulesolution is attached to a tip of a pin 13; at (4) the moving pin 14 iswashed in a washing section 15, n^(th) DNA is removed, and thereafter,n+1^(th) DNA is attached to the moving pin 14. Returning to FIG. 3,1^(st) to K^(th) DNAs are attached one after another to the pins 13 on apin drum 16, being spaced at specific intervals.

The attached DNAs are then attached to the substrate 1 one after anotheras the pin drum 16 is rotated. The DNAs 3 are thus placed on thesubstrate 1. Assuming that a half of the minimum DNA interval is definedas t, FIG. 3 illustrates that DNAs are spaced by intervals 1t, 2t, 3t,and 5t.

Immobilization of DNA in the attached DNA microcapsule 6 and thesub-solution 8, i.e., immobilization of capture DNA onto the substrate1, will be described with reference to FIG. 7. As shown in FIG. 7(1),the DNA microcapsule 6 and the sub-solution 4 are attached onto thesubstrate 1. The vaporization temperature of sub-solution 4 is lowerthan the melting point of a membrane 6. Therefore, when the temperatureis slightly increased at (2), the sub-solution 4 is evaporated, leavingonly the microcapsule 6. When the temperature is further increased at(3) to the melting point of the membrane 6, the membrane 6 is melted sothat the fluid of the melted membrane 6, the main solution 4, and thecapture DNA 3 are mixed to a solution. In this case, the membrane 6 maybe made of a material whose vaporization temperature is lower than thevaporization temperature of the main solution and the membrane 6 may beevaporated. A surface of the substrate 1 has been subjected to surfacetreatment so that DNA is easily immobilized on the surface. Therefore,at (4) the capture DNA 3 is immobilized on the substrate 1 as shown inFIG. 8. A part or the whole of the main solution 4 is dried at (5) andwashed at (6), thereby completing the DNA spot 2.

In the present invention, arrangement of DNA spots 2 a, 2 b, and 2 a ismodulated so as to incorporate positional information thereinto.According to this positional information, the positional orders of therespective DNA spots 2 can be determined. At the same time, as shown inFIG. 5, a DNA spot region 17 and a data region 18 are separated fromeach other. In the data region, a substrate ID 19, a DNA numbercorrespondence table 20 for the position of a DNA spot and a DNA spotID, and the sequence data 21 of DNA itself (biomolecule attribute data)(data structure shown in FIG. 5(2)) are modulated and recorded in a dotpattern. The dot pattern can be read by an XY scanner. Therefore, thearrangement data of DNA spots can be read by an XY scanner.Alternatively, data in the data region can be read out using anexcitation laser for allowing a sample to generate fluorescence. FIG. 6shows a specific example of DNA substrate attribute data. DNA substrateID 19, a DNA number-position correspondence table 20 indicating thecorrespondence between DNA numbers and positional information, and DNAsequence data 21 indicating the DNA sequence of each DNA, has beenrecorded as data in the data region before shipment from the factory.Note that the DNA sequence data of a DNA number is encrypted using anencryption key and then recorded. Personal DNA data is information of ahigh level of personal privacy which has to be stringently protected,and therefore, is encrypted using a public key, such as RSA, ellipsecode, or the like, or a high-bit encryption key. Therefore, even if aDNA chip or a DNA substrate containing subject information is run off,the DNA sequence of a specific DNA spot cannot be read without anencryption key. Therefore, personal DNA information can be preventedfrom leaking. It is also conceivable to accumulate DNA sequence data 21in a DNA management center without recording the data in DNA chips inorder to improve security. The user informs a DNA management center of aDNA substrate ID 19 and a reaction between labeled DNA 22 and a DNA spot2, i.e., fluorescence level data. Next, the center searches a DNAsubstrate database for the DNA sequence of each DNA spot using the DNAsubstrate ID 19. The center further analyzes a reaction result of a DNAspot and labeled DNA 22, DNA sequence-disease correspondence data todiagnose and predict a disease (in the case of a human), and sends onlynecessary information to laboratory medical technologist or a doctor inencrypted form. With this system, privacy information is prevented fromimproperly leaking.

Example 2 Fabrication Example of Biomolecule Chip (2): Ink Jet Method

The pin spot method has been described above. Next, a method ofattaching DNA to a substrate using ink jet will be described. FIG. 9 isa block diagram showing an ink jet (Bubble Jets) attaching apparatus. Anink jet nozzle 24 contains microcapsules 9 a, 9 b containing DNA andempty microcapsules 23 a, 23 b, 23 c, 23 d containing only main solution4, which are supplied from an ink supplying section 25. A specific emptycapsule contains a specific dye for indicating address information. Amaster control section 28 sends an eject command to an eject signalgeneration section 29 and then an eject control circuit 27. As a result,an eject section 26 generates heat so that bubbles occur. The bubblescause the microcapsule 9 a to be ejected toward a substrate. The emptymicrocapsules 23 b, 23 c are ejected. However, the empty microcapsules23 b, 23 a are unnecessary. When a photodetector 31 detects an emptymicrocapsule, a removal signal generation section 30 sends a removalsignal to an unnecessary liquid removing section 32. In the unnecessaryliquid removing section 32, a deviation field is applied to a deviationsection 33 so that unnecessary liquid in the empty microcapsule 23 isremoved as indicated by dashed-line arrow 34 and does not reach asubstrate.

The photodetector 31 has color filters 31 g, 31 h, 31 i (R, G, B, etc.),and therefore, can detect the color information of an emptymicrocapsule. The photodetector 31 also has a counter section 111. Afirst counter 111 a counts the number of microcapsule blocks. A secondcounter 111 b counts the number of DNA microcapsules. A third counter111 c counts the number of empty microcapsules. When there are fourcolors, 2-bit address data is obtained from a set of emptymicrocapsules. 16-bit address data is obtained from 8 emptymicrocapsules. When two bit of the 16 bits are used as check bits, it ispossible to precisely check if the order, arrangement, or number ofmicrocapsules is incorrect. Therefore, incorrect attachment can beadvantageously prevented. Even when microcapsules are not colored, 2bits can be obtained by ejecting 1, 2, 3, or 4 microcapsulesconsecutively. When 8 sets are used, 16 bits can be obtained, i.e., thesame size of address data as above can be obtained. The addressinformation of a microcapsule obtained by the photodetector 31 is sentvia an address output section 31 p to the master control section. DNAnumber can be identified based on address information. For example, asshown in step 68 m in a flowchart of FIG. 15, if a microcapsule makesthe number of DNA capsules having DNA number n greater by one, a removalsection described below removes the microcapsule.

On the other hand, the substrate 1 is moved by a predetermined amount bya shift section 37 controlled by a shift amount control circuit 36 basedon a signal from a shift amount detector 35, so that DNA spots 2 a to 2h are attached onto the substrate 1 as shown in FIG. 10(4).

Next, the flowchart of FIG. 15 will be described. Initially, step 68 asets m=0, n=1. If a synchronization capsule sequence is detected at step68 b, a capsule block number m of the first counter 111 a is increasedby one in step 68 c. Whether or not m is the last number is checked instep 68 d. The arrangement and order of empty microcapsules in m^(th)block are checked in step 68 f. If a result of step 68 g is incorrect,the process goes to step 68 m. If the number of microcapsules is greaterby L than a reference number, an eject signal (=1) is output in step 68n for L capsules. A removal signal (=1) is output so as to remove acapsule. This operation is carried out L times to cause the arrangementto be normal and the process then returns to step 68 g. If a result ofstep 68 m is NO, the process goes to step 68 p. If the number ofmicrocapsules is smaller by L than the reference value, ejection isstopped for clocks corresponding to L microcapsules in step 68 q. Duringthis time, DNA spot 2 is missing. Therefore, a defect block flag is setto be 1, which is recorded in the data region 18 on the DNA substrate 2,indicating the presence of the defect. The address of a microcapsule inan address counter 112 or an address block counter 113 is corrected byincreasing the address by L.

Now, the process returns to step 68 g. The number of DNA microcapsulesis checked in step 68 h. If the result is OK, the eject signal is set tobe ON and the removal signal is set to be OFF for one unit in step 68 i.In this case, microcapsules are ejected so that one DNA spot is formed.In this case, it is judged that there is no defect, and the address fora DNA microcapsule is increased by one(step 68 k). The process thenreturns to step 68 b. Thus, DNA spots 2, which contain correspondingDNA, can be formed.

Now, an ejection procedure using ink jet will be described withreference to FIG. 11. In (1), (2), the microcapsule 9 a containingn^(th) DNA reaches the tip of the nozzle 24. When a voltage is appliedto the eject section 26 in (2), a bubble is generated so that themicrocapsule 9 a is ejected in (3) and is attached to the substrate 1 in(4). This method is called BUBBLE JET® ink jet. A piezoelectric devicemay be provided instead of the eject section 26 to obtain the sameeffect. In this case, by applying an eject voltage to the piezoelectricdevice, a microcapsule is ejected in a piezoelectric ink jet method.Meanwhile, the microcapsule 9 b containing (n+1)^(th) DNA reaches thetip portion. When an eject voltage is applied in (5), the microcapsule 9b is ejected toward the substrate 1. In (6), (n+2)^(th) microcapsule 9 cis transported to the tip portion. In this case, the empty microcapsules23 d, 23 e among the three consecutive empty microcapsules 23 a, 23 d,23 e indicating a synchronization mark are present on the photodetectors31 a, 31 b. These empty microcapsules have a high level oftransmittance, both of which are detected by the photodetector 31. Thecapsules are detected as synchronization marks. Therefore, themicrocapsule 9 d following these capsules is recognized as containing(n+3)^(th) DNA from a correspondence table. Therefore, it is possible toprevent ejection of DNA having an erroneous number due to displacementof a microcapsule. A synchronization mark is composed of 2, 3, or 4empty capsules. One set can contain 2-bit data. A synchronizationcapsule is used to match DNA itself to its DNA number, so that thematched DNA can be ejected. If DNA having a specific number is notejected as in step 68 p of FIG. 15, lack information is recorded in thedata region of FIG. 5. As shown in FIG. 12, for example, a plurality of(n+1)^(th) DNA spots are formed as DNA spots 3 a, 3 b, 3 c. Therefore, alack of DNA spot does notcause a problem. In (7), the synchronizationcapsules 23 d, 23 e reach the tip portion. These capsules do not containDNA and are unnecessary. Therefore, a removal signal is applied in (8),the capsules are deviated and removed by the unnecessary liquid removingsection 32 so that the capsules do not reach the substrate 1. Theremoval circuit can prevent unnecessary substances from being attachedto the substrate 1.

A light detection signal, an eject signal, a removal signal and anarrangement of DNA spots will be described with reference to FIG. 10.Initially, a system is operated in accordance with a shift clock in FIG.10(3). Initially, since a DNA capsule has a low level of lighttransmittance as shown in FIG. (1), the DNA capsule is detected insynchronization with an eject signal in (4). Synchronization capsulesare detected as shown in (2) since the two photodetectors 31 a, 31 b areboth turned ON. As shown in (4), an eject signal is generated both for amicrocapsule containing DNA and a synchronization capsule containing noDNA. However, a removal signal of (5) is generated in synchronizationwith an eject signal following a detection signal of (2) so that allsynchronization capsules are removed. In the present invention, in orderto specify the DNA number of each DNA spot 2, positional data, such asaddress or the like, is buried as the intervals between DNA spots asshown in (11). When positional data is 12 bits in length, the positionaldata is divided into 00, 01, 10, 00, and 01 as shown in (10), where 00corresponds to a mark interval of 3 clocks and 01, 10 and 11 correspondsto 4t, 5t and 6t, respectively. Thus, interval modulation is performed.Further, there is a synchronization mark 37 having an interval of lot.With this method, positional information can be buried when DNA spotsare formed. Since the address of each DNA spot is obtained, the DNAnumber of a first DNA spot for a synchronization mark is obtained fromDNA number positional information 20 shown in FIG. 6. In the presentinvention, therefore, the DNA numbers of all DNA spots 2 can beidentified. The sequence information of all DNA spots can be obtained byusing sequence information 21 for DNA numbers recorded in the substrate1 of FIG. 6. Since the address is obtained, absolute precision is notrequired when the position of a DNA spot is read out. Therefore, ahigh-precision XY scanner is not required and it is possible to prepareand read a DNA spot with a low-precision apparatus, thereby making itpossible to supply an inexpensive DNA testing apparatus. In the priorart, ultrahigh-precision fabrication and readout apparatuses arerequired in order to increase the density of DNA spots. In the presentinvention, high density can be achieved by a low-precision fabricationand readout apparatuses. Further, since the attribute data of DNA spotsare recorded on the same substrate 1 as shown in FIG. 6, the possibilityof incorrectly reading out a DNA attribute is advantageously eliminated.

FIG. 30 shows a sub-nozzle 116, a sub-eject section 114, and asub-solution supply section 115 in addition to the system of FIG. 9. Thesub-nozzle 116 is supplied with microcapsules, and a sub-solution fromthe sub-solution supply section 115.

Operations will be described from (1) to (6) in sequence. In (1), (2)and (3), a DNA capsule 9 a is transported, and in (4), is ejected. In(5), a large volume of sub-solution is released from the sub-solutionsupply section 115 and then removed by the removal section 32. In (6), aDNA microcapsule 9 b is transported. In this method, the inside of thenozzle is washed with the sub-solution, whereby the mixing of DNA can beprevented.

Example 3 Fabrication Example of Biomolecule Chip (3): Tube Method

Next, a specific method for fabricating a biomolecule chip according tothe present invention and a configuration thereof will be describedwhere a fiber convergence system is described as an example. Note thatalthough in Example 3 a fiber convergence type fabrication method isused as an example, a method for burying data (e.g., address, chip ID,and the like) by arrangement of biomolecule spots, which is a feature ofthe present invention, can be applied to other methods, such as a PINmethod, an ink jet method, a semiconductor masking method, and the like.

In this method, initially, a probe 131 corresponding to a specific DNA,RNA and protein is injected together with a gel solution into a hollowthread tube 130 from a container 132 containing the probe 131 in the gelform. Different probes 131 a, 131 b, 131 a, 131 d, etc. are injectedinto respective tubes 130 a, 130 b, 130 c, 131 c, 130 d, etc., which arethen bundled in an X direction, i.e., horizontally, to form a sheet 133as shown in FIG. 33( b). Further, the bundled sheets 133 a, 133 b arepiled so that the tubes 130 are arranged in a matrix to form a block 137as shown in FIG. 33( c). The tubes may be arranged in a circle to form acolumn-like block.

In one embodiment of the present invention, a mark tube 134 for a markindicating an address or data is placed in the block. Note that in asecond method, a mark tube 136 is placed in the block, which comprises aprobe solution 135 or a tube 130 in which a material for reflecting,absorbing, or emitting fluorescence having a specific wavelength iscontained. The mark tube 136 will be described in detail below. AlthoughFIG. 33( c) shows a 10×10 matrix, the actual matrix has a side ofseveral hundreds to several thousands of tubes.

The block 137 is sliced in a Z direction, so that a chip 138 iscompleted. The chip 138 is fixed on a fix plate 139. The fix plate 139is used to fix a chip and may comprise a container for holding aspecimen, and is shipped in this form. The fix plate 139 is used withoutmodification to perform testing. On the fix plate, a fix plate ID 140,which varies depending on corresponding attributes of probes, isrecorded in the form of a bar code, characters, or a bit pattern. A chipID for managing a process control or the attribute data of a chip isrecorded in the first sheet 133 a by a method for burying data accordingto the present invention. This attribute data can be used to identifychips having different probe sequences. Therefore, by checking, it ispossible to detect when an incorrect fix plate ID 140 is provided to achip 138.

(Method for Burying Data)

A spot of each probe is placed on the chip 138. Data is buried in anarrangement of spots. A spot containing the probe 131 for detecting DNAor protein is herein referred to as a DNA spot 2. Such a spot may alsobe called biomolecule spot. Now, a method for burying data will bedescribed. Specifically, as shown in FIG. 35(3), for example, 10biomolecule spots 141 e to 141 p are aligned in an x direction. Two markspots 142 a, 142 b are placed at the left end and three mark spots 142c, 142 d, 142 e are placed at the right end. Firstly, the case where themark spots include no biomolecule will be described. The case where themark spots include a biomolecule(s) will be described later.

A mark spot has an optical property different from a biomolecule spot141 in terms of specific wavelength. Specifically, a mark spot has areflectance or absorbance with respect to a specific wavelength, or thepresence or absence of fluorescence or the intensity of fluorescencewith respect to a specific wavelength, different from a biomoleculespot. Therefore, a mark spot can be clearly distinguished from thebiomolecule spot 141. For example, when there is a difference inreflection or absorption with respect to excited light or irradiation,the mark spot can be optically clearly distinguished from thebiomolecule spot as indicated by hatched lines in FIG. 35(3). The sameeffect can also be obtained when the wavelength of fluorescence of themark spot 142 with respect to excited light is different from thewavelength of fluorescence of the biomolecule spot 141. The intensity ofreflected light or fluorescence obtained by irradiating the mark spotwith light having a specific wavelength is illustrated in FIG. 35(4). Agroup of the mark spots 142 in (3) are collectively calledidentification mark 143. 2-bit codes, “10”, “11”, and the like areassigned to respective identification marks 143 e, 143 f. FIG. 35(2)shows a general view including the identification marks 143 e, 143 fwhere part of biomolecule spots 141 between identification marks areomitted. This figure shows 7 identification marks 143 a to 143 g, towhich codes 00, 10, 11, 01, 10, 11, and 00 are assigned, respectively.When the identification marks 143 a, 143 g containing 4 consecutive markspots 142 are used as synchronization marks 144 a, 144 b, the fiveidentification marks 143 between the synchronization marks 144 can beused to bury 10-bit data, i.e., 10, 11, 01, 10, and 11.

By reading these marks with a scanning beam or a CCD in a testapparatus, the 10-bit data is read from this region. If these 10 bitsare used as, for example, address data, the address of this region,“1011011011”, can be obtained only by reading the 10 bits. Thus, a thirdbiomolecule spot 141 x to the right of the identification mark 143 c is23^(rd) biomolecule spot in address “1011011011”. Therefore, theidentification number 145 of a biomolecule spot can be identified as“101101011-23”. Therefore, all biomolecule spots 141 on a chip can beindividually identified without counting spots from an end of a matrix.Thus, a conventional method for identifying a spot by counting spotsfrom an end of a matrix to the x, y coordinates of the matrixcorresponding to that spot, is made unnecessary.

By reading out the attribute information of a biomolecule spot 141corresponding to the identification number 145 from an attribute table146 in FIG. 40, various tests and diagnoses can be made possible. Theattribute information contained in the attribute table 146 of FIG. 40includes the sequence or genetic information, or marker information of aspecific disease, of a biomolecule having this identification number, orDNA, RNA, or other substances hybridizable to this biomolecule probe,and the like.

In an actual fabrication method, for example, a tube piling method,errors in piling are accumulated, so that it is less likely that the xand y coordinate axes of a matrix can be precisely formed. In this case,therefore, the identification number of each biomolecule spot identifiedfrom such x and y coordinates is highly likely to match the correctidentification number. An incorrect identification number leads to anincorrect test result. In the case of a DNA test or the like, when anincorrect test result is used for diagnosis of a patient, falsediagnosis occurs frequently, potentially causing a serious problem.

In contrast, the present invention has an advantageous effect that theidentification number of a biomolecule 141 can be precisely identifiedby reading locally the vicinity of the biomolecule 141 even ifbiomolecule spots are not arranged in a precise matrix. Biomolecule chipfabrication methods other than a semiconductor process can be used tofabricate a large number of chips containing biomolecule spots. Even inthe case of a perfect matrix arrangement obtained by a semiconductorprocess, as the number of spots is increased, error occurs in countingspots on a test apparatus, potentially resulting in an incorrectidentification number. In the present invention, it is not necessary tocount spots with respect to x and y from an end of a biomolecule chip,and therefore, counting error does not occur. Moreover, theidentification number of each biomolecule spot can be obtained byreading only the vicinity of the biomolecule spot, whereby theidentification number of a desired biomolecule spot can be identified ina short time.

Specific procedures are as follows. For example, it is assumed that DNA,RNA, or the like with a label emitting fluorescence with a wavelength ofλ2 has been hybridized to a biomolecule spot 141 x. When the biomoleculespot 141 x is irradiated with excited light having a wavelength of λ1,the biomolecule spot 141 x emitting fluorescence with a wavelength of λ2can be observed. According to the data burying method of the presentinvention, the identification number of the biomolecule spot 141 x isobtained and the sequence of the DNA or the like can be obtained from anattribute table, thereby making it possible to analyze or testspecimens.

Example 4 Test Using Biomolecule Chip

(Test Procedures)

Procedures for test or diagnosis will be described with reference to aflow chart shown in FIG. 41. Initially, in step 148 a, a specimen with afluorescent label is provided and hybridized to a surface of abiomolecule chip 138 fabricated by a chip fabrication process, such as atube method, a semiconductor process method, an ink jet method, a PINmethod, or the like. Unhybridized specimens, which are unnecessary, areremoved in step 148 b. This chip is loaded into a laser scanning typetest apparatus or a CCD readout type test apparatus shown in FIG. 14described later. In step 148 c, chip ID written in the first line of thechip 138 or/and a fix plate ID 140 on the fix plate 139 of FIG. 33 isread out by a light beam, CCD, or the like, so as to check the chip IDor the fix plate ID against a predetermined one. Next, these IDs arechecked against a predetermined ID list. If a result of theabove-described check is incorrect, the process is stopped. If theresult of the check is correct, an attribute table 146 (see FIG. 40)corresponding to the chip ID is obtained via a network 150, such as theInternet, LAN, or the like, and is temporarily stored in a memory 151 ofa test apparatus 149.

The process goes to a test mode. In step 148 d, m is set to be 0. Instep 148 e, m is increased by one. In step 148 f, a surface of the chipis irradiated with excited light having a first wavelength of λ1. Whilescanning the chip using a laser or a CCD, a wavelength separationfilter, such as mirrors 65, 66 in FIG. 14, is used to search for m^(th)biomolecule spot emitting fluorescence with a specific wavelength. Thesearch is continued until the spot is found in step 148 g. When them^(th) biomolecule spot is found, the chip is irradiated with excitedlight or reference light in step 148 h. An optical property of a markspot 142, such as reflectance or the like, with respect to thewavelength of excited light is set to be different from that of abiomolecule spot 141. Therefore, as shown in FIG. 35(2), the mark spotcan be optically distinguished from the biomolecule spot using excitedlight or reference light. In this case, the same effect is obtained evenwhen the mark spot 142 emits fluorescence having a wavelength differentfrom that of the biomolecule spot 141. Therefore, the mark spot 142 canbe detected. In step 148 j, n is set to be 0. In step 148 k, n isincremented by 1, the code of n^(th) identification mark 143 isidentified. When the process is repeated until n reaches the last n instep 148 n, one data row is obtained. This data row contains an errorcorrection code 152 in order to improve the reliability of the data row.Therefore, the data row is subjected to error correction in step 148 p,and the data row having no error is obtained in step 148 q. In step 148r, by counting the number of spots from an identification mark 143closest to a subject biomolecule spot, the total number of biomoleculespots from a synchronization mark to the subject biomolecule spot 141(e.g., 141 x) as shown in FIG. 35(2) can be obtained. In step 148 s, theidentification number 145 of the m^(th) biomolecule spot is identifiedbased on the address and the number of counters. In this case, thewavelength of fluorescence can be specified from a filter setting, andtherefore, the label number of the fluorescence is identified.

In step 148 t, the attribute table 146 corresponding to chip ID is readout from the memory 151, and the sequence data of DNA or the like havinga specific identification number is retrieved as shown in FIG. 40.Therefore, the type of DNA, RNA, or a protein contained in a specimencan be obtained. In step 148 u, this information and the identificationnumber are registered in to a test database 147 of the memory 151. Instep 148 v, it is checked whether any other biomolecule spots emittingfluorescence remain unread. If there is an unread spot, the processreturns to step 148 e and the biomolecule spot emitting fluorescence islocated. If there is no unread spot, data of test database 147 is sentto an analysis program 155 in step 148×. Instep 148 y, an analyzed testor diagnosis result is output. Thus, the operation is completed.

As described above, according to the present invention, data, such asaddresses, chip ID, or the like, is buried in the arrangement ofbiomolecule spots. Therefore, the identification number of a biomoleculespot of interest can be obtained from the arrangement of biomoleculespots or mark spots around the identification number of interest. Thisdata can contain chip ID and chip attribute data as well as addresses.In this case, all data required for testing or analysis is obtained froma chip itself. If chip ID obtained from a chip is compared with the fixplate ID 140 of the above-described fix plate, incorrect fix plate IDcan be checked in testing, thereby reducing the occurrence of erroneousdetection due to incorrect fix plate ID caused by a mistake in amanufacturing process. Further, it is possible to distribute abiomolecule chip alone without a fix plate 139, whereby chip cost can bereduced.

Note that, for the sake of simplicity, as shown in FIG. 35, the examplein which the biomolecule spot 141 containing biomolecules and the markspot 142 without a biomolecule, i.e., two types of spots, are used tobury data, such as addresses, has been first described. In this method,a mark spot is only added, and therefore, it is easy to managefabrication. On the other hand, the density of biomolecule spots isdisadvantageously reduced. For applications requiring a higherbiomolecule spot density, as shown in FIG. 34( a′), a mark solution 153having optical properties, such as a reflectance, absorbance, andrefractive index with respect to a specific wavelength, fluorescence,and the like, different from the solution 135 in (a) is introduced intoa tube. This tube is disposed as is a tube 130 a in (b). As shown in(d), a mark biomolecule spot 154 is formed on a chip. When it is notefficient to prepare two biomolecule solutions, i.e., one with a markand one without a mark, a mark may be attached to a tube 130 asrepresented by mark tubes 134, 136. In this case, although thesensitivity of such a mark is reduced, substantially the same effect asthat of a mark biomolecule spot 154 described in FIG. 36 is obtained.

This fabrication method can be applied to other applications. In thecase of a PIN method, a mark material is added to a main solution 4 or asub-solution 8 as shown in FIG. 2 to prepare a mark solution 153. Asshown in FIG. 38, biomolecules in a normal solution indicated by anunfilled circle and in a mark solution indicated by a filled circle arefixed to the substrate 1. Therefore, biomolecule spots 141 a to 141 iand the mark biomolecule spot 154 containing the mark solution 153 canbe formed on the substrate 1 as shown in FIG. 38. The description ofFIG. 38 is omitted because of substantially the same operation as inFIG. 3.

In the case of an ink jet method, a mark microcapsule 156 containingbiomolecules and a mark solution is loaded instead of thesynchronization capsules 23 d, 23 e shown in FIG. 11 so as to attach thecapsule 156 onto the substrate 1 as shown in FIG. 39(4), (5), (6), and(7). Thereby, the same arrangement of the biomolecule spot 141 and themark biomolecule spot 154 as in FIG. 38 can be obtained. Moreover, whena semiconductor mask is used, the same effect is obtained by piling amaterial for a mark on a mark biomolecule spot.

In the above-described three fabrication methods, the arrangement of thebiomolecule spots 141 and the mark biomolecule spots 154 on the chipsubstrate is the same as in FIG. 36. The same effect as in the tubemethod can be obtained when the mark spot 142 is used instead of themark biomolecule spot 154 as shown in FIG. 35. In this case, a solutionor material containing only a mark solution without a biomolecule isimmobilized on a chip substrate in the three fabrication methods. Thefour exemplary fabrication methods have been described. The presentinvention can be applied to various biomolecule chip fabrication methodsother than the four examples.

Returning to FIG. 35, another data burying method will be described.FIGS. 35(2) and (3) show that the mark spots 142 are arranged so thatdata, such as addresses or the like, is buried, where the number ofconsecutive mark spots is in the range of 1 to n. Hereinafter, adescription in parentheses corresponds to FIG. 41. FIG. 36(5) shows thatdata is buried by changing the interval between the mark spot 142 (markbiomolecule spot 154) depending on the data. Specifically, two markspots having an interval corresponding to 8 biomolecule spots is definedas a synchronization mark 157. The intervals between mark spots 142(mark biomolecule spots 154) from the synchronization mark 157 a to 157b are 4, 5, 6, 7 and 4. Therefore, data corresponding to 5 digits in theseptinary number system, i.e., 7 to the power of 5 pieces of data, canbe buried. This data can contain address data, error correction code,and chip attribute data. In this case, the mark spot can be easilydetected since the mark spot has the longest interval.

FIG. 35 (FIG. 36) (6) shows a method in which two consecutive mark spots142 (mark biomolecule spots 154) are arranged as a synchronization mark158. In this case, the intervals between mark spots 142 (markbiomolecule spots 154) from a synchronization mark 158 a to 158 b are 3,6, 4, 5, and 8. Therefore, 7 to the power of 5 pieces of data can beburied.

FIG. 37( a) shows an arrangement of long biomolecule spots 161 a, 161 band 161 c, and biomolecule spots 141 a to 141 k, which is obtained by atube method using a flat tube 160. When the elongated biomolecule spot161 a is regarded as one mark spot, two contiguous elongated biomoleculespots 161 a, 161 b can be defined as a synchronization mark 162. Similarto the synchronization mark 158 in FIG. 35(6), 7 data is read from anarrangement of 7 biomolecule spots 141 k, 141 j in FIG. 37. Thus, asshown in FIG. 37( b), “75456”, i.e., 5-digit data in the octanary numbersystem can be buried between the elongated biomolecule spot 161 b andthe subsequent elongated biomolecule spot 161 (not shown).

In the present invention, a method for correcting an error in data usingerror correction codes is adopted. In the case of 10 bits, as indicatedin a data structure diagram in FIG. 42, 10-bit original data 159 (A₀ toA₉) is provided with 2-bit error correction codes 152 (C₀, C₁) producedfrom the original data 159 using Reed Solomon coding or turbo coding,thereby correcting an error. Therefore, the reliability of buried datais increased, so that an error is unlikely to occur in important data,such as addresses. In FIG. 42, an error correction code is used only inthe horizontal direction. When a product code method in which an errorcorrection code is additionally used in the vertical direction, it takesa longer time to perform operations and obtain original data. However,in this case, there liability of buried data is improved.

Example 4 Detection Apparatus for Biomolecule Chip

In the above-described manner, a DNA chip or a DNA substrate, on whichcapture DNA is arranged, can be fabricated. This DNA substrate can beused to test DNA or a protein.

DNA, such as cDNA or the like, is extracted from a DNA specimen, and islabeled with a fluorescence material 38 to prepare labeled DNA 22. Asshown in FIG. 13(1), the labeled DNA 22 is applied to a DNA substrate ofthe present invention. The DNA substrate is placed under specificconditions, such as heating at several degrees Celsius and the like, tocarry out hybridization. As shown in FIG. 13(2), the labeled DNA 22 iscoupled with capture DNA 3 a in the n^(th) DNA spot.

Now, a method for detecting this labeled DNA or a labeled protein usinga DNA substrate of the present invention will be described. FIG. 14 is ablock diagram showing a detection apparatus 39 for detection. Firstly,the left half of the block diagram will be described. Light emitted froman excitation light source 40, such as laser or the like, is convergedby a mirror 41 having wavelength selectivity and a lens 42, and isfocused on a substrate 1. Reflected light from the substrate 1 reaches adetection section 43 via the mirror 41 and a polarizing mirror 42. Afocus error signal detection section 45 sends a focus error and atracking error to a focus control circuit 46 and a tracking controlcircuit 47, respectively. An actuator 48 drives and controls the lens 42in such a manner as to match the focus with tracking. A focus offsetsignal generation section 49 and a track offset signal generationsection 50 apply an offset to the focus and the tracking in order tooptimize a label detection signal. In the present invention, thearrangement of DNA spots 2 is intentionally modulated to includepositional information. A procedure for reproducing this data will bedescribed below.

A main signal is reproduced by a main signal reproduction section 69. Apositional information detection section 64 detects positionalinformation. A track number output section 52 and a DNA spot numberoutput section 51 send a currently scanned DNA spot number and tracknumber to a data processing section 55. Thereby, a DNA spot isidentified.

A signal from a data region 18 shown in FIG. 5 is reproduced to data bya demodulation section, such as EFM, PM, or the like, in the main signalreproduction section. The data is subjected to error correction in anECC decoder 53. DNA substrate attribute data shown in FIG. 6 isreproduced by a DNA substrate attribute data reading section 54, and issent to the data processing section 55. In the data processing section55, the capture DNA identification number 58 of a currently scanned DNAspot 2 a is identified. A mirror 65 is used to send fluorescencecorresponding to the capture DNA identification number 58 to a firstlabel signal detection section 60. Therefore, the label intensity data(fluorescence level, etc.) of a first identified DNA having a specificidentification number corresponding to the capture DNA 3 is output fromthe label signal output section.

A fluorescence dye 38 of the first labeled DNA 22 linked to the DNA spot2 a is irradiated with excited light from a light source 40 having afirst wavelength λ0. After the emission of fluorescence is started andcontinued for the half life, the fluorescence comes to a half level. Thehalf life is in the range of several nanoseconds to several tens ofmicroseconds.

FIG. 17(1) shows the output power of excited light. FIG. 17(2) shows theintensity of fluorescence emitted from a fluorescent material or afluorescence dye by excited light. FIG. 17(2) shows that thefluorescence intensity comes to the half life at t=t6.

Now, a method for separating wavelengths will be described in detailwith reference to FIG. 16. Of a plurality of incident light beams λ0, λ1and λ2, the excited light with wavelength λ0 having the highestintensity is reflected by the mirror 41 having an optical film 68 a witha film thickness of λ0/4. Fluorescence with wavelength λ1 from a firstlabel is reflected by the mirror 65 having an optical film 68 b with athickness of λ1/4, while fluorescence with wavelength λ2 transmits themirror 65. Thus, the three wavelengths are separated. The transmittanceof the separated wavelength is less than or equal to 1/1000, andtherefore, the crosstalk between each wavelength is suppressed.Therefore, a weak fluorescence label level can be detected. A λ/4 filterfor λ0 can be added to further improve the degree of separation, therebymaking it possible to suppress the components of the excited lightsource 40, and therefore, increasing the S/N ratio. As shown in FIG. 18,an excited light beam 71 is made smaller than the DNA spot 2. Forexample, the size of the excited light beam 71 is as small as severalmicrometers. In this case, the DNA spot 2 can be divided in a scanningdirection, i.e., a direction of a scanning track 72. The resultantportions are called cells 70 a, 70 b, 70 c, 70 d. Four pieces of dataare obtained in the scanning direction, thereby making it possible tomeasure the distribution of the amount of fluorescence with higherprecision. Cells 70 g, 70 h are measured as follows: a track errorsignal, which is V0 in the track offset signal generation section 50, isintentionally generated; the track error signal is input to the trackingcontrol circuit 47; an offset is generated in a track direction; and asindicated by a scanning track 72 a in FIG. 18, a track is shifted sothat the cells 70 g, 70 h are scanned while applying excited light tothe cells 70 g, 70 h. When an inverse track error signal is input, cells70 e, 70 f can be scanned. Thus, in the case of FIG. 18, the DNA spot 2is divided into 8 cells which can be scanned and irradiated with excitedlight.

Next, a detection procedure will be described with reference to FIG. 20.FIG. 20(1) shows that DNA spots 2 a to 2 i are arranged on a DNAsubstrate 1. In (2), first labeled DNA 22 a having a label with a firstfluorescence wavelength λ1 and second labeled DNA 22 b having a labelwith a second fluorescence wavelength λ2 are introduced onto a substrateto carry out hybridization. The first labeled DNA 22 a iscomplementarily coupled with DNA contained in DNA spots 2 b, 2 e, 2 h.The second labeled DNA 22 b is coupled with a DNA spot 2 h. In (3),drying is performed, and scanning is started using an excited lightsource 40 with a wavelength λ0. (4) shows an excited light detectionsignal for reflected light of the excited light. Fluorescence generatedby the excited light with the wavelength λ0 does not contain awavelength component of λ0, so that only a detection signal with λ0 isobtained. The surface of the substrate 1, such as glass or the like, hasa certain reflectance. Nevertheless, a reflection layer may be furtherformed on the surface of the substrate of FIG. 1 in order to increasethe signal level of the excited light detection signal. A detectionsignal as shown in (4) is obtained due to the difference in reflectancebetween the reflectance of a DNA spot 2 with respect to λ0 and thesurface of the substrate 1. As described concerning the procedure forDNA spot formation with reference to FIG. 10, positional data or thelike is buried by changing the pattern or arrangement of DNA on thesubstrate 1 of the present invention depending on specific data. Asshown in (4), the interval between detection signals varies. As aresult, signals 00, 01, 10, 00, 01, 01 can be reproduced as shown in(5). Based on these signals, positional data, i.e., address informationas shown in (6) can be reproduced. Therefore, for example, it can befound that a DNA mark 2 a is located in 260^(th) track and at 1128^(th)address. A detection apparatus obtains DNA substrate attribute data fromthe data region 18 of the substrate 1 as described with reference toFIG. 6. Specifically, for example, the start number of DNAidentification number in 260^(th) track in DNA number positionalinformation 20 is 243142. Therefore, DNA having identification number244270 can be identified. Further, when the user can obtain anencryption key 73, encrypted DNA sequence information for DNA number(=244270) in sequence information 21 by DNA number is decoded by acipher decoder 74. Thereby, the DNA spot 2 can be identified to theextent that the DNA spot 2 has a DNA sequence ATCTAGTA . . . Note thatwhen the user does not have the encryption key 73, DNA sequence is notdecoded. In this case, even if the fluorescence data of a DNA spot isobtained, the privacy of personal DNA information is protected. In apostscript data region 76 of FIG. 6, first label attribute data 77 andsecond label attribute data 78 of hybridized labeled DNA is additionallyrecorded, such as the excited light wavelengths 410 nm, 410 nm oflabels, fluorescence wavelengths 700 nm, 600 nm, half lives 100 ns, 100ns, or the like. Therefore, the operations of the detection apparatuscan be checked or set using this postscript data.

Returning to FIG. 20, a procedure for measuring the fluorescence of alabel generated by excited light will be described. Initially, asdescribed in FIG. 18, in the case of the scanning track 72, cells 70 a,70 b, 70 c, 70 d are scanned with excited beam 71. In the case of a DNAspot 2 b, fluorescence with a wavelength λ1 is generated, and isdetected by the first label signal detection section (FIG. 14). As aresult, a first label detection signal 85 a corresponding to the fourcells are detected as shown in (8). When a DNA spot 2 g is scanned,fluorescence with a wavelength λ2 is generated, and a second labeldetection signal 85 b is generated as shown in (9). When an offset isapplied in FIG. 18, i.e., in the case of the scanning track 72 a, alabel detection signal 85 c resulting from detection of fluorescenceonly from two cells is obtained as shown in (10).

In the present invention, when a higher detection sensitivity of labeledDNA is required, the excited light source 40 is caused to emitintermittently. A shift amount in a linear direction or a rotationaldirection of the substrate is detected by a shift amount detector 86. Apulsed light emission signal 88 or a sub-pulsed light emission signal 87having reversed phase is generated by the pulsed light emission controlsection 87 depending on the shift amount. In first scanning, as shown in(11), when the pulsed light emission signal 88 is applied to the lightsource 40, pulsed light emission is performed. As a result, first andthird cells, i.e., cells 70 a, 70 c, generate fluorescence. In thismethod, fluorescence is detected when the light source 40 is in the OFFstate. Therefore, a considerably high SIN is obtained. For example, alabel detection signal 85 d is obtained as shown in (13). In this case,a light receiving portion of the first label detection section isslightly shifted, so that light receiving efficiency is improved. Insecond, i.e., even numbered, scanning, a sub-pulsed light emissionsignal having reversed phase as shown in (12) is input to the lightsource 40, and the same track 72 is scanned. Due to the reversed phasewith respect to the first scanning, second and fourth cells, i.e., cells70 b and 70 d (two clocks after) (FIG. 18) are irradiated with excitedlight. During the subsequent on clock, excited light is OFF. Therefore,fluorescence from the cell 70 b or 70 d can be detected withoutinterference by excited light. Thus, by scanning two times, thefluorescence levels of all cells can be advantageously detected withhigh sensitivity. A description will be given with reference to aflowchart of FIG. 31. In step 118 a, scanning is performed once so thatthe arrangement information of all DNA spots on the track is stored intoa memory (steps 118 b, 118 c). In this case, in step 118 d, if scanningis performed at a constant speed in the second time or thereafter, thepositions of the DNA spots can be reproduced by reading out from thememory (step 118 f).

A description will be given with reference to FIGS. 31 and 32. In step118 g, excited light is intermittently emitted at odd numbered clocktimes (FIG. 32(2)). Fluorescence is generated (FIG. 32(4)). In step 118h, fluorescence is detected based on a detection permitting signal ofFIG. 32(3) (FIG. 32(5)).

In third scanning, excited light is intermittently emitted at evennumbered clock times in step 118 j (FIG. 32(6)). In step 118 k,fluorescence is intermittently detected (FIG. 32(9)). Therefore, theinfluence of excited light is eliminated, whereby SN is improved.

Accordingly, in the present invention, high precision in the lineardirection is obtained even by pulsed light emission. No problem arisesin precision in the track direction.

Next, a method for improving sensitivity while enhancing positionalresolution will be described. Referring to FIG. 19, the substrate 1 ismoved so that the cell 70 b is moved in the order of (1), (2), (3), (4),(5), and (6). In order to measure the amount of fluorescence from thesubstrate 1, a light detection section 90 has an array structure 91 andperform switching in the order of (1′), (2′), (3′), (4′), (5′), and(6′), depending on the shift amount. Thereby, a high level ofsensitivity is obtained while keeping a resolution. FIG. 21 is a blockdiagram showing that switching is performed on the array by a switchingsection 92 based on a signal from the shift amount detector 87 and asynchronization signal from the DNA spot 2; the fluorescence of the cell70 b is tracked, and fluorescence data is accumulated and output by anaddition section 93. In this case, if the amount of shift from thecenter of a cell is within f×0.05 where f represents the focal distanceof the lens 42, the cell can be detected by the array 91.

A label detection signal list 94 in the detection apparatus has data asshown in FIG. 22. This data is recorded into the data region 18 of FIG.5 with excited light. In this case, all data can be integrated with asingle substrate. Therefore, the possibility of obtaining incorrect datais eliminated, thereby avoiding accidents, such as false diagnosis andthe like.

FIG. 23 shows that a recording layer 95 is added to a substrate 1 and alight source 40 and a lens 42 a are provided on the opposite side of thesubstrate to the recording layer 95. Since data can be recorded in therecording layer 95, a large volume of data can be recorded. FIG. 24shows that two upper and two lower actuators 48, 48 a are mechanicallycoupled together. In this case, as shown in FIG. 25, since the positionof each DNA spot 2 a can be defined with an address 96 in the recordinglayer 95, the outer shape of the DNA spot 2 a can be specified with astart address 97, an end address 98, innermost circumferential tracknumber 99, and an outermost circumferential track number 100, therebymaking it possible to access a DNA spot at high speed. Thiscorrespondence list can be recorded in the recording layer 95. Referringto FIG. 26, DNA spots 2 a, 2 b, 2 a are in the shape of a rectangle,thereby making it possible to perform scanning tracking with higheraccuracy. FIGS. 27 and 28 shows DNA chips as shown in FIG. 5 but in theshape of a circle. In particular, the entire rear surface of the DNAchip of FIG. 28 can be used. Therefore, the DNA chip of FIG. 28 has alarge recording capacity and can store the entirety of a DNA sequence.The term DNA is herein used. Any biomolecule as defined herein (e.g., aprotein) may be used as a subject substance to be labeled. RNA may beused instead of DNA. Cells or a part of tissue may be used as long asthey can be arranged on a substrate.

In the embodiments, as a method for fabricating a DNA substrate, a PINmethod and an ink jet method are employed to describe the examples ofthe present invention. However, the present invention can also beapplied to a semiconductor process method. Referring to FIG. 29, in asemiconductor process method, probe DNA is arranged on a glasssubstrate. Referring to FIG. 29(2), lithography is performed as follows.A mask 120 is used to perform masking. Specific probe DNA is irradiatedto activate an elongation reaction probe DNA containing A (adenine) 123is formed as shown in FIG. 29(3). Thereafter, C (cytosine) 124 is formedas shown in FIG. 29(4) and (5). This elongation reaction is carried outfor A, C, G, and T, i.e., four times, to complete one layer. If theelongation reaction is carried out 4N times as shown in FIG. 29(6),probe DNAs having a length of N bases are formed. In the DNA chipfabrication of the semiconductor process method, the position of theopening of the masking is shifted as shown in FIG. 10(7) using thepresent invention. As shown in FIG. 29(1), a mask 121 a is shifted withrespect to the original mask 121 b in correspondence with specific data.Thereby, positional data can be buried and recorded in an arrangement ofDNA spots.

Example 6 Network Type Test Apparatus

An operation of a test system using a biomolecule chip according to thepresent invention will be described. FIG. 43 is a flowchart showing theoperation of the test system of the present invention. In a biomoleculeextraction section 172, a biomolecule is extracted, purified, or grownfrom a sample 171 collected from a subject 170 to prepare a specimen173. In a test section 175 of a main test system 174, this specimen 173is loaded to a biomolecule chip 138, followed by a reaction. A portionof molecules in the specimen 173 are hybridized with probes in aspecific biomolecule spot 141 as described above. This biomolecule spotexhibits label information, such as fluorescence or the like, andtherefore, can be easily detected. On the other hand, a chip ID 19 canbe detected from the biomolecule chip 138. These test data are encryptedtogether with the chip ID 19 (substrate ID 19 is also referred to aschip ID 19), and the encrypted data is sent via a communication section176 and then through the Internet 177 or a communication circuit to acommunication section 179 of a sub-test system 178, such as a testcenter or the like. Thereafter, the data is sent to an analysis section181 of an analysis system 180. In the analysis section 181, theattribute data of each biomolecule spot of a corresponding chip ID canbe obtained from an identification number-attribute database 184. Fromthe obtained attribute data and label number, the state of the specimen173, such as a gene, a protein or the like, can be identified.

An analysis result in the case of genetic information is shown in FIG.45. Firstly, a gene number 183 corresponding to a gene sequence isshown. Gene attribute data indicating a gene attribute 184 of a genecorresponding to the number contains the sequence of the gene; a markerfor a disease, a character, or the like; and the like. Since this typeof test is used in a test of a specific disease or a test of a specificmolecule, the main test system 174 outputs a request, such as,specifically for example, data “Please output information relating to adisease a”. As shown in a selective output 185 of FIG. 45, onlyinformation relating to a request output 186 is selected by a selectionsection 182, and is encrypted by the output section 183 and sent via thecommunication section 179 and the Internet 187 to the main test system174.

In a gene test, data which is not originally intended is obtained in thecourse of testing and analysis. For example, when genetic information ona specific cancer is required, if unintended genetic information, suchas other diseases or characters (e.g., an intractable and unavoidabledisease (juvenile Alzheimer's disease, etc.), a catastrophic character,etc.), is output, the interest of a subject is likely to be damaged. Ifthis type of information is unintentionally leaked, a privacy problemoccurs. According to the present invention, the selection section 182filters out information unrelated to a request output or raw geneticinformation, thereby making it possible to prevent unnecessaryinformation from being output.

The result of a test corresponding to a chip ID, which is requested tothe main test system 174, is received by the diagnosis system 187 andprocessed by a diagnosis section 188. The chip ID-subject correspondencedatabase 191 can be used to identify the subject 170 from a chip ID 19.All chips have a unique chip ID. Therefore, the subject corresponding toeach chip can be identified. This data is not sent to any sub-testsystem. Therefore, patient data is prevented from being leaked to a testlaboratory or the outside of a hospital. The test system can know therelationship between a subject and a chip ID, but does not have theattribute data of each biomolecule spot of the chip ID. Unless theattribute data is obtained from the sub-test system, whole geneticinformation cannot be obtained. In other words, the main test system 174and the sub-test system 178 each have incomplete complementaryinformation, thereby maintaining security. Thus, the security of thegenetic information of a subject can be protected.

In this case, each chip ID is different from the others and is providedwith a randomized number. Therefore, even if all the attributeinformation of a chip having a certain chip ID number (e.g., theattribute data of a biomolecule corresponding to the identificationnumber in each biomolecule spot) is made public, the data of any otherchip ID cannot be identified, since there is no correlation between thespecific chip ID and the other chip IDs. The security of the wholesystem can be protected as long as the secrecy of the sub-test systemcan be maintained. When the secrecy of the main test system ismaintained, no information connecting a chip to a person is obtainedeven if a chip and a personal ID are obtained by a third party. In thiscase, security is further improved.

The diagnosis section 188 outputs a result of diagnosis based onhistoric data of a subject (a disease, etc.) and a test result obtainedfrom the sub-test system. A diagnosis result output section 192externally outputs the diagnosis result. A treatment policy productionsection 189 produces a plurality of treatment policies based on thediagnosis result, assigns priorities to the treatment policies, andoutputs the treatment policies through the output section 190.

(Utilization of Genetic Information Other than Diseases)

In the above-described examples, information relating to a specificdisease is specified as request information and is sent out as a requestoutput 186. Recently, it has been revealed that a psychologicalattribute, such as a character or the like, of a subject can be obtainedfrom genetic information. For example, a person having the third exon ofthe dopamine D4 receptor on the 11^(th) chromosome has a character ofchallenge. Thus, now and thereafter, attribute information, such as apersonal character, will be clarified from genetic information one afteranother. Considering this point, attribute data indicating apsychological feature, such as a character, of a subject is added to thedisease data in the request output 186 of FIG. 43. In this case,information on the character, aptitude, or the like of a subject 170 issent from a sub-test system to the diagnosis system 187. The prioritiesof the treatment policy options of the treatment policy productionsection 189 are changed depending on the attribute and aptitude data ofthe subject. For example, for a subject who prefers a high risk and ahigh result, the priority of a treatment option which provides a highresult at a high risk is increased. For a subject who prefers a moderateresult at a low risk, the priority of a therapeutic agent, which is safebut whose effect is not high, is increased. With this method, adiagnosis system for providing a treatment policy suitable for thecharacter or aptitude of a subject can be achieved.

Example 7 Stand-alone Test Apparatus

The operation (security, etc.) of the present invention has beendescribed using the exemplary network type test apparatus of FIG. 43.The present invention can be applied to a stand-alone type test system193 as shown in FIG. 44.

The network type test system of FIG. 43 comprises two systems, i.e., amain test system and a sub-test system. The latter is administered by aneutral entity, such as a test center, and can enhance secrecy tomaintain the security of the whole system. In contrast, the stand-alonetype test system of FIG. 44 includes a black box section 194 having ahigh level of secrecy instead of the sub-test system 178. The black boxsection 194 leaks no internal information other than informationrequired to be output to the outside. Only required data is output froman input/output section 195. The security of information is maintainedby the black box section 194.

Most portions of the stand-alone type test apparatus have the sameoperation as in FIG. 43. Only different points than those in FIG. 43will be described below. Initially, in a test section 175, encrypteddata, such as encrypted biomolecule spot-attribute data 146 encryptedusing public key encryption function or the like, is reproduced by achip 138 and is sent to a black box section 194. This encrypted data isdecoded to plain text data by a cipher decoding section 197 within theblack box section 194. This plain text data contains attributeinformation relating to each biomolecule spot on a biomolecule chip. Theattribute information is added to a biomolecule spot identificationnumber-attribute database 184.

In the case of FIG. 43, the main test system accesses via a network tothe database 184 in a sub-test system, such as a test center or the likeand obtains data. Therefore, such a sub-test system, such as a testcenter or the like, needs to obtain the latest data of all chipsproduced all over the world and update the data as occasion demands. Themain test system cannot obtain a test result unless a network isavailable. However, in the method of FIG. 44, even if a chip is recentlyproduced, the attribute data 146 is present in the biomolecule chip, andthis attribute information is automatically recorded into theidentification number-attribute data correspondence database 184, whichis thus updated, every time a chip is loaded in a test system.Therefore, the stand-alone type test system does not have to beconnected to a network. Moreover, a memory of the test system storesdata corresponding to only one chip. Therefore, memory capacity can besignificantly reduced. In the case of this method, a mobile type testapparatus can be used. Similar to FIG. 43, in this method, onlyinformation relating to an output requested from the main test system isselected by a selection section 182, and is sent from the black boxsection 194 to a diagnosis system 187 in the main test system 174.

Note that if the black box section 194 is produced in such a mannerthat, for example, the black box section 194 is incorporated into a chipof LSI and its external terminals are limited to the input/outputsection 195 and the cipher decoding section 197, no internal data can beexternally read out. Therefore, security is protected. As describedabove, with a biomolecule chip containing encrypted data of the presentinvention and a stand-alone type test system of the present invention,required testing or diagnosis can be carried out without a network orexternally input data while protecting the information security of asubject.

Note that although the above-described example is such that theattribute information of a biomolecule chip is buried in the arrangementdata of biomolecule spots, such information may be optically recordedwith pit marks or the like on a substrate integrated with a chip asshown in FIG. 5. Alternatively, as shown in FIG. 46, a biomolecule chip138, an IC chip 198 having a non-volatile memory 201, and an electrode199 are provided on a substrate 200, the attribute information may berecorded in the non-volatile memory 201 of the IC chip 198. Theattribute information may be optically read out in the test system, ormay be electrically read out from the electrode 199 or the like.

All of the publications, patents, and patent literature cited herein areeach incorporated herein by specific reference. The present inventionhas been described with reference to various particular and preferableembodiments and techniques. However, it should be understood thatvarious modifications and variations can be made without departing fromthe spirit and scope of the present invention.

Note that in the description of the embodiments, the arrangement ofbiomolecule spots is changed in the same direction as a single specificarrangement direction of biomolecule spots. However, other methods canbe easily implemented, though their descriptions are omitted. First ofall, the size of a biomolecule spot may be changed. Specifically, data“01” is assigned to a biomolecule spot having a small size; “10” isassigned to a biomolecule spot having a middle size; and “11” isassigned to a large size. Thus, three-valued data can be buried in onebiomolecule spot.

Alternatively, the position of a biomolecule spot may be intentionallyshifted from a reference position in a direction perpendicular to aspecific arrangement direction of biomolecule spots. Specifically, data“01” is assigned to a biomolecule spot shifted by +20% with reference tothe reference position; “10” is assigned to a biomolecule spot shiftedby 0%, i.e., not shifted; and “11” is assigned to a biomolecule spotshifted by −20%. In this case, three-valued data can be buried in onebiomolecule spot. If the number of the shift amounts or resolutions isincreased, multivalued data, such as five-valued data, seven-valueddata, or the like, can be buried.

Alternatively, the size of a biomolecule spot may be changed in adirection perpendicular to a specific arrangement direction without theposition of the biomolecule spot. For example, data “0” is assigned toan elliptic biomolecule spot having a major axis in the verticaldirection, and data “1” is assigned to a circular biomolecule spot,thereby making it possible to bury two-valued data. Alternatively, thesize of a biomolecule spot may be changed in the same direction as thearrangement direction.

If a plurality of methods of the above-described burying methods aresimultaneously used, the amount of buried data can be further increased.

INDUSTRIAL APPLICABILITY

As described above, in the present invention, the position or patternitself of a biomolecule (e.g., DNA, RNA, a protein, a low weightmolecule, etc.) is changed to bury the positional information of thebiomolecule. Therefore, no extra process is required and conventionalhigh-precision positioning is no longer required. This method is moreeffective when the number of types of biomolecule is large and thedensity of biomolecules is required. Further, a test apparatus can readout the positional information of a DNA spot using an excited lightsource, and therefore, biomolecule spots may be only relativelypositioned. No conventional high-precision apparatus for absolutelypositioning biomolecule spots is required. Thus, a test apparatus can beobtained by only a simple configuration. Furthermore, data is recordedon a substrate, and the data is read out using excited light. Therefore,the attribute data of a biomolecule spot can be read out from the samesubstrate without increasing the number of components, whereby datamatching error is eliminated. The above-described advantageous effectsaccelerates widespread use of a biological test apparatus and diagnosisapparatus.

1. A method for fabricating a biomolecule substrate, comprising thesteps of: 1) providing a set of biomolecules and a substrate; 2)preparing biomolecule microcapsules by i) producing, from a mainsolution, an emulsion comprising a medium and micro-emulsion particlescontaining an intended biomolecule from the set of biomolecules on thebiomolecule-type-by-biomolecule-type basis and ii) producing a polymerthin film by interfacial polycondensation at an interface between themicro-emulsion particles and the medium so that the micro-emulsionparticles are covered with the polymer thin film, thereby obtaining thebiomolecule microcapsules; 3) dispersing the biomolecule microcapsulesinto a sub-solution; 4) spraying the sub-solution containing thebiomolecule microcapsules onto the substrate; and 5) setting thetemperature of the sub-solution containing the biomolecule microcapsulesduring spraying to be higher than the melting point of the polymer thinfilm.
 2. A method according to claim 1, further comprising the step ofwashing the biomolecule microcapsules after the preparing step.
 3. Amethod according to claim 1, wherein the spraying step is performed byan ink jet method.
 4. A method according to claim 3, wherein the ink jetmethod is performed by a BUBBLE JET® ink jet method.
 5. A methodaccording to claim 1, wherein the microcapsules of the set ofbiomolecules of different types are disposed at different positions. 6.A method according to claim 1, wherein the spraying step is performed bya PIN method.
 7. A method according to claim 1, wherein the biomoleculeis selected from the group consisting of DNA, RNA, a peptide, andcomplex molecules thereof.
 8. A method according to claim 1, wherein thebiomolecule is DNA.
 9. A method according to claim 1, wherein thebiomolecule is cDNA or genomic DNA.
 10. A method according to claim 1,further comprising the step of performing labeling specific to eachbiomolecule microcapsule.
 11. A method according to claim 1, wherein thepolymer thin film has a thickness of 10 to 20 μm.
 12. A method accordingto claim 1, wherein the intended biomolecule is dissolved and dispersedin a water phase.
 13. A method according to claim 1, wherein thevaporization temperature of the sub-solution is lower than the meltingtemperature of the polymer thin film.
 14. A method according to claim 1,wherein the vaporization temperature of the polymer thin film is lowerthan the vaporization temperature of the main solution.